TTHHÈÈSSEE

En vue de l'obtention du

DDOOCCTTOORRAATT DDEE LL’’UUNNIIVVEERRSSIITTÉÉ DDEE TTOOUULLOOUUSSEE

Délivré par l'Université Toulouse III - Paul Sabatier

Discipline ou spécialité : Biologie moléculaire, cellulaire et du développement

JURY

Jean Antoine Lepesant (DR) Elzbieta Pyza (Prof.)

Catherine Soula (Prof.) Alain Vincent (DR)

Zbigniew Dabrowski (Prof.) Maria Slomczynska (dr hab.)

Ecole doctorale : Biologie, Santé, Développement

Unité de recherche : Centre de Biologie du Développement Directeur(s) de Thèse : Alain Vincent et Zbigniew Dabrowski

Rapporteurs : dans le Jury

Présentée et soutenue par Joanna Krzemien Le 27 octobre 2008

Titre : "Control of larval hematopoiesis in Drosophila; microenvironment, precursors and

cell lineage".

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“Control of larval hematopoiesis in Drosophila;

microenvironment, precursors and cell lineage".

Joanna Krzemień

PhD thesis made in “co-tutelle”

at Jagiellonian University in Cracow

and Université Paul Sabatier in Toulouse

2008

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Acknowledgements

Taking adventage from the opportunity that gives me this manuscript, I would like to

thank to everyone who has helped me to live the experience of PhD studies. It is a very

difficult task, which I will probably not fulfil perfectly. Sometimes though, even a smile from a

person that You’ve just met, can give You plenty of joy and force to go further.

These four years of my PhD were rich in experience and interesting rencontres. I’ve

met many people who more or less had impact on my life. I’ve learnt plenty of things about

the surrounding world. I had the chance to have excellent guides at the scientific tracks and

wonderful company to enjoy my time. There were a lot of people, who, even if not directly

involved in the scientific part of my PhD, made it possible for me to study in Toulouse and in

the same time, stay attached to my Jagiellonian University in Krakow.

Without too much introduction, I would like simply and very sincerely to thank:

to Tomek Skalski, who was actually a key person at the beginning of my PhD. He contacted

me with Alain Vincent and (see below ;) and encouraged me a lot in my choices.

to My Dear Profesor Zbigniew Dąbrowski, who let me free, who helped me at each step of my

international experience, who was always there when I needed a piece of advice, a joke or

just to talk; who was my Friend and Teacher.

to Alain Vincent for plenty of things; first of all for adopting me into CBD family, then for

discussions, ideas, jokes, corrections, all the time that he spent for his students, among them

lucky Joanna; for patience and formation; for support and enthusiasm. Thank You!

to Michele Crozatier a master of flies, for guiding me in the world of Drosophila and fly

hematopoiesis, for teaching me so many things! For being always there, when I need her, for

tiramisu, honey when I had a throat ache and for taking care of me ☺

to Rami, a “brother in PhD” for all the patience that he had for me ☺, for all knowledge that

he shared with me, for discussions not always very scientific ;) for giving me the confidence.

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to Delphine, for staying with us, for making me laughing, for together dissecting, for nice

time that we have had with Maeva and Gaylord who were funny, who were laughing and who

were always there for me when I needed a good company.

to Nico for the joy that he spreads all around, for always good mood, for big heart and all

parties that we enjoyed together

to Hadi for making me smile, for supporting polish canoeing for having fun in the lab and for

being a good man

to Monika, Bruno, Carmen, Virginie, Jonathan, Justine people who accompanied me in the

laboratory life. Each of them completely different made me learn a lot about respect for

others and about the joy of life.

to Brice, Julian, Phiphi, Serge, Christian, Eric, Yannick, Daniel, Mohamad, Dani, Thomas,

Alex, my boys, for smiles, nice words, bisous, parties, ambiance and actually, just for being

there ☺

to Brice and Aurelie for showing me world through the microscope, for patience, help and

experience that I got thanks to them and to Bruno, for coping with complicated informatic

problems and curing my computer

to Murielle, Elea, Aisha, Gaelle, Gwenaelle, Helene, Cathy, and Aurelie for them, simply

to Mylene, Elizabeth and Philippe for taking care of me ☺

to all my Friends and colleagues from CBD for four years, which I spent in Toulouse and

even if sometimes I had difficult moments, I could always, find someone to smile to me ☺

to prof. Marian Lewandowski and doc. Maria Slomczynska, who made it possible for me to

get through the meanders of administration and exams; always friendly and helpful

to Monika, my polish Friend, discovered in Toulouse, who was my Small Poland in France,

who made me feel like at home

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to my Polish Friends Sławek, Kama, Kasia, Kamila, Monika, Paweł, Mariusz and Kuba (Your

names and all which I haven’t written here, are written in my heart) for being my joy!; for all

hello and goodbye parties that I could always count on when I was coming back home. for

Your support and warmth that helped me live far away from home and for making out me a

smiley person.

to my Parents, my Brother and the whole Family who were always with me even being far

away

to Laurence, for everything, that I cannot enclose in words…

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Abbreviation list:

A1 - A8 - abdominal segments ael - after egg laying AGM – aorta - gonad – mesonephros AMPs - antimicrobial proteins Antp - Antennapedia Bam - Bag of marbles Bc - Black cells BMP – Bone Morphogenetic Protein BrdU - bromodeoxyuridine cc – crystal cells Cg25C - Collagen25C CNS - central nervous system COE - Collier/Olfactory-1/Early B cell Factor Col - Collier CRQ - crocquemort CXC - C-X-C chemokine Cyc - Cyclin Cyo - Curly of Oster CZ - cortical zone DBD - DNA binding domain Dome - Domeless DoxA3 - Diphenol oxidase A3 Dpp - Decapentaplegic Dscam - Down syndrome cell adhesion molecule dSR – CI – Drosophila scavanger receptor EBF – Early B-cell factor ECM - extracellular matrix EH - embryonic hemocytes EL - egg length ESC - escorting stem cell Esg - Escargot fbn - facial branchiomotor neurons Flp - Flipase FOG - Friend of GATA FRT - Flipase Recombination Targets GAT - gastrointestinal associated lymphoid tissue Gbb - Glass-bottomed boat Gcm - Glial cells missing GFP – green fluorescent protein GMC – ganglion mother cell GSC - germline stem cell H3P - phosphorylated Histone 3 Hh - Hedgehog HLH - helix-loop-helix Ho - Hoechst Hop - Hopscotch Hop Tum-l – Hopscotch Tumorous-lethal Hs - heat shock HSC - hematopoietic stem cell Ig - immunoglobulin Imd - immune deficiency IP - intermediate progenitors

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IPT - Ig-like domain shared by Plexin and Transcription factor ISC - intestine stem cells JAK/STAT - Janus Kinase/Signal Transducer and Activators of Transcription L1, L2, L3 – firt, second, third instar larvae LG - lymph gland Lin- - lack of lineage differentiation markers Lz - Lozange MHC - Major Histocompatibility Complex MZ - medullary zone N - Notch NB - neuroblast Neur - Neuralised NimC1 - Nimrod NK - natural killer cell Odd – Odd skipped PDGF - Platelet-derived growth factor PGRP - Peptidoglycan recognition protein PNS - peripheral nervous system PO - Phenoloxidase Pon - Partner of Numb proPO - Prophenoloxidase PRRs - pattern recognition receptors Prx – Peroxidasin PSC – posterior signalling center Pvf - PDGF- and VEGF-related factor Pvr - Drosophila homolog of receptor for cytokines of platelet-derived growth factor (PDGF) family and vascular endothelial growth factor (VEGF) receptor RGC - radial gliall cells RNAi – RNA interference Rpr –Reaper S1 – signal 1 S2 – signal 2 Sca - stem cell antigen SCZ - subcortical zone Ser - Serrate Shh – Sonic hedgehog SNSD - self non-self discrimination SOP - sensory organ precursor cells Sp - Sternopleural Srp - Serpent SSC - somatic stem cells SVZ - subventrical zone T1-T3 – thoracic segments Tep - Thioester-containing protein UAS - Upstream Activating Sequence Upd - Unpaired Ush - U-shaped VEGF - Vascular Endothelial Growth Factor Vkg - Viking VLP - virus like particles W - White Wg - Wingless Wnt – Wint wt – wild type Y-Yellow

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Contents

Introduction…………………………………………………………………....13

The Immune system in Metazoa ........................................................................................................ 15

Hematopoiesis in Drosophila melanogaster..................................................................................... 19

The Drosophila immune response..................................................................................................... 19

The Drosophila hemocytes................................................................................................................ 23 Plasmatocytes.............................................................................................................................................. 23 Crystal cells ................................................................................................................................................. 27 Lamellocytes................................................................................................................................................ 29

Embryonic hematopoiesis ................................................................................................................. 31

Larval hematopoiesis ........................................................................................................................ 33

Control of larval hematopoiesis........................................................................................................ 43

Collier, a transcription factor with multiple developmental functions ........................................... 45

Osteoblasts, hematopoietic stem cells and their niche...................................................................... 49

The hematopoietic stem cells ............................................................................................................ 53

Drosophila melanogaster stem cell niches as a model for vertebrate systems. ................................ 57

Hematopoietic stem cells in Drosophila? ........................................................................................ 63

Materials, methods and experimental design…………………………………65

Antibodies and reagents.................................................................................................................... 67

Fly strains ......................................................................................................................................... 67

BrdU pulse-chase experiments.......................................................................................................... 69

Mitotic index measurements.............................................................................................................. 69

Notch ts experiment............................................................................................................................ 71

Clonal analysis.................................................................................................................................. 71

Results………………………………………………………………………….75

Introduction....................................................................................................................................... 77

The PSC is required to maintain a pool of prohemocytes in 3rd instar larvae................................. 77

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Ser expression in PSC cells is involved in maintenance of col expression. ...................................... 79

The JAK/STAT pathway is activated in the MZ ................................................................................ 81

Wasp infestation switches off JAK/STAT signalling in the MZ and induces massive differentiation of

prohemocytes. ................................................................................................................................... 83

PSC cells extend numerous filopodia............................................................................................... 85

Looking for stem cells. ...................................................................................................................... 87

Waves of mitosis during development and after wasp infestation .................................................... 95

Cell lineage analyses ........................................................................................................................ 99

Lamellocytes and crystal cells share a common progenitor ........................................................... 103

Notch signalling and cell fate restriction........................................................................................ 107

The extracellular matrix of the lymph gland................................................................................... 109

Discussion…………………………………………………………………….111

The PSC as a model of hematopoietic niche................................................................................... 113

Collier expression in the lymph gland, a matter of golden mean. .................................................. 119

Role of the PSC in immune response against the parasitoid wasps................................................ 119

Are there hematopoietic stem cells in the lymph gland?................................................................. 123

Proliferation versus differentiation................................................................................................. 127

The origin of larval hemocytes....................................................................................................... 131

What more could we learn with clonal analysis? ........................................................................... 135

Concluding remarks........................................................................................................................ 137

References…………………………………………………………………….141

Appendix……………………………………………………………………...155

Article.............................................................................................................................................. 155

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Introduction

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The immune system in Metazoa

All Metazoans, the multicellular animals, are capable to keep self-integrity by recognising

“non-self’ tissue thanks to their immune system. The immune system must make specific

distinctions so that it can eliminate the pathogen without destroying the body’s own tissues. In

the “self non-self discrimination” (SNSD) theory the recognition receptors are central to

immunity, however it seems not to be sufficient. Actually, it is very important to recognise

what poses harm to the body among self and non-self tissue, in order to avoid the detrimental

reaction against foreign but non-dangerous agent, like for example food, commensal bacteria

or just naturally changing self-tissue. The “theory of Danger” proposed by Matzinger suggests

that damage rather then “foreignness” is what initiates an immune response. In this model the

alarm signal is released from the body’s own cells and triggers the immune reaction (Janeway

1992; Matzinger 1994; Kvell, Cooper et al. 2007).

Two branches of the immune response have evolved in the Metazoans: innate and adaptive

immunity. Innate immunity is germline encoded and considered as non-specific,

nonanticipatory, nonclonal, whereas the adaptive response is specific, anticipatory, clonal and

somatic (rewiewed in (Kvell, Cooper et al. 2007). Innate immunity is shared by all metazoans

and can be grouped into cellular (that uses specific cells as, e.g., phagocytes) and humoral

(mediated by secreted factors, like antimicrobial peptides) compartments. A fundamental

experiment, showing the cellular aspects of innate immunity, was made in 1882 by

Metchnikov. Upon inserting a horn into the body of a starfish larva he could observe that

many mononuclear cells were “attacking” and engulfing the horn in a process that he termed

phagocytosis. This finding brought him a Nobel price in 1908 (Petranyi 2002).

Immune-related innate functions like phagocytosis or cytokine secretion were already

developed in pre-bilaterian times, 700 million years ago, in sponges and higher aquatic

invertebrates. The innate immune response, although very efficient lacks, the memory

function that appeared only with vertebrates. Around 500 milion years ago, primitive fishes

developed jaws and thanks to this invention got access to a true predatory life, enjoying great

advantage over agnathas. However, they also began to be subjected to a higher rate of

infections by microbes and parasites in their gastrointestinal tract. The infections were the

result of increased physical injuries caused by swallowed animals containing scales and

skeletal elements. Additionally new prey–predator relationships created new food chains,

through which microbes, e.g. viruses, could spread crossing the species barriers.

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Fig. 1. Phylogenetic tree of the animal kingdom highlighting the evolution of key immunological elements. Innate immunity operates in the entire animal kingdom. Adaptive immunity appeared with jawed vertebrates about 500 million years ago (green frame) (after Kvell et al.. 2007). The position of Drosophila in the animal kingdom is marked by a red frame.

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The development of jaws was followed by establishment of a new immune system, called

gastrointestinal associated lymphoid tissue (GAT), containing T-lymphocyte-like cells.

Generation of the complex repertoire of immune receptors in lymphocytes through somatic

gene rearrangement emerged 350-400 million years ago and the system antigen presenting

proteins called MHC (major histocompatibility complex) appeared around 300 milion years

ago. This resulted in development of adaptive immune response characterised by a remaining

memory that permits a shorter and stronger secondary response thanks to the clonal expension

of activated lymphocytes (Matsunaga and Rahman 1998; Petranyi 2002; Kvell, Cooper et al.

2007).

Figure 1 shows the phylogenetic tree of animal kingdom underlining the evolution of key

immunological elements.

Millions of invertebrate species rely uniquely on the innate immunity and only around

45 000 today vertebrate species use also an adaptive response. It is postulated that the

generally small size of invertebrates does not allow them to come up with the large number of

cells required for operation of the adaptive immune system. Not only the number of cells but

also the number of cell types (complexity), raised during the evolution of Metazoa are highest

in vertebrates. Only vertebrates could afford to run such a costly system without the risk that

the costs would outweigh benefits (Klein 1997; Kvell, Cooper et al. 2007). Needless to say,

invertebrates cope very well with immune challenge and innate immunity is efficient for their

vital needs. Since innate immune responses are both relatively simple and many mechanisms

are highly conserved during evolution, invertebrates have become the subject of very

extensive studies.

One of the best described model organisms is Drosophila melanogaster, called a fruit fly.

Drosophila is a holometabola insect whose life cycle is composed of the embryonic stage,

three larval stages and a pupal stage from which the imago finally emerges. Drosophila

spends most of its life cycle in decaying organic matter such as rotting fruit, an environment

enriched in microorganisms, that constantly challenges the immune system (Tzou, De

Gregorio et al. 2002).

In Drosophila melanogaster (as in all other invertebrates), specific cells called hemocytes,

the analogues of vertebrate blood cells, are key players in the response to immune challenges.

These cells are formed during fly development in a process called hematopoiesis. During my

PhD work, I studied the control of this complex process at cellular and molecular levels.

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A B C

Fig. 2. The three types of Drosophila hemocytes (A) Crystal cell containing crystals of proPhenylOxidase; (B) Plasmatocyte, the macrophage-

like phagocyte; (C) Lamellocyte, the encapsulating hemocyte.

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Hematopoiesis in Drosophila melanogaster

Hematopoiesis of Drosophila melanogaster, thanks to the great interest that this model

organism has received for many years, starts to be very well described. Currently, we know

that it occurs in two spatially and temporally distinct phases (Evans, Hartenstein et al. 2003;

Holz, Bossinger et al. 2003; Meister and Lagueux 2003). The first phase, which is often

compared to the vertebrate primitive hematopoiesis, takes place during embryogenesis in the

head mesoderm. The second phase takes place during the larval stages, in a specific

mesodermal organ, the lymph gland. It has been proposed to represent a model for the

vertebrate wave of definitive blood formation. As a result of hematopoiesis, three hemocyte

types form in Drosophila: plasmatocytes and crystal cells that are produced both in embryo

and larvae and lamellocytes which are specific of the larval stages (Fig.2 A-C) (Meister and

Lagueux 2003). In contrast, to vertebrate blood cells, Drosophila hemocytes participate in

neither the transport of oxygen, since there exists a tracheal system which supplies the gazes

to each body area, nor the nutrition or hormone transport, because the hemolymph circulation

system is open. The main hemocyte tasks are to perform efficient immune responses and

assure the proper development of the organism.

The Drosophila immune response

As for probably all metazoans, including vertebrates, the immune system of Drosophila

is based on two main branches: the humoral response and the cellular response. The humoral

response is the secretion, mainly by fat body and epithelia, of antimicrobial proteins (AMPs)

that degrade the invader and other immune effectors (ex. reactive oxygen species, opsonins,

clotting factors). Epithelia are the first barrier against the invading microorganisms and

successfully protect the animal from the infection. The fat body is the major immune-

responsive tissue. Thanks to its large size and broad localization, it is an efficient organ for

the secretion of peptides into the hemolymph where they reach easily the proper

concentration. The fat body is believed to be somewhat analogous to the mammalian liver

(Lemaitre and Hoffmann 2007). The humoral response allows protection against a broad

range of bacteria, fungi and viruses. Infection by Gram positive or Gram negative bacteria and

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++

Antimicrobial peptidesopsonins (Teps), clotting factorsstress factors

Bacteria Gram+Fungi

Bac teria Gram-

A B

Tollpathway

TollPGRP-LC

Imdpathway

JAK/STATpathway

Domeless

Fat body

Fig. 3. Schematic overview of Drosophila host defenses. Detection of pathogens induces specific immune responses in specialised tissues depending on the type of pathogen, via activation of various signalling pathways (after Lemaitre and Hoffmann 2007, modified).

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fungi induces different patterns of AMP expression in the fat body, suggesting that the

microbial recognition mechanism can discriminate between these various classes of invaders

(Lemaitre, Reichhart et al. 1997). Recognition of microorganisms is a crucial step for the

efficient immune response and it involves the series of proteins known as pattern recognition

receptors (PRRs) (that can be soluble or membrane bound) and recognise microbial molecules

(Wang and Ligoxygakis 2006). The recognition of pathogen can lead to activation of specific

signalling pathways: Fungi and Gram+ bacteria trigger the Toll signalling pathway, Gram

negative bacteria lead to Imd (immune deficiency) pathway activation and some viruses

engage the JAK/STAT signalling pathway (Fig. 3A) (Lemaitre and Hoffmann 2007).

The massive production of AMPs that occurs following infection is primarly regulated at

the transcriptional level. Characterisation of promoters of inducible immune genes revealed

that ĸB-like recognition motifs were involved in the activation of AMPs. The three

Drosophila members of transcription factor ĸB (NF- ĸB/Rel) family: Dorsal , Dif and Relish

were shown to contribute to the response to infection and signal through Toll and Imd

pathway by translocating to the nucleus and act as the switch of AMPs expression upon

infection (Engstrom, Kadalayil et al. 1993; Han and Ip 1999; Stoven, Ando et al. 2000; Uvell

and Engstrom 2007). Some AMPs are very stable and are still detected in hemolymph several

weeks after immune challenge (Uttenweiler-Joseph, Moniatte et al. 1998).

The JAK/STAT signalling pathway has been also proposed to be involved in the fat body

humoral response, where it triggers the expression of the complement-like protein tep1 and a

stress gene turandotA of unknown function. JAK/STAT deficient flies are however resistant

to bacterial and fungal infection and they show normal AMP profile (Lagueux, Perrodou et al.

2000; Ekengren and Hultmark 2001; Ekengren, Tryselius et al. 2001; Ekengren, Tryselius et

al. 2001; Agaisse and Perrimon 2004; Lemaitre and Hoffmann 2007).

The humoral response has been extensively studied and is a subject of many excellent

recent reviews (Ferrandon, Imler et al. 2007; Lemaitre and Hoffmann 2007; Uvell and

Engstrom 2007). Although it was believed that insects were devoid of the immune specificity

and memory, last few years have brought evidence that they could exist in Drosophila

(Watson, Puttmann-Holgado et al. 2005; Pham 2007). Possibly the future will bring us more

surprises in this area.

The second aspect of the immune response, that is the cellular aspect, is less well known.

Cellular immune responses rely upon three types of hemocytes that participate in three

main immune processes: plasmatocytes are engaged in phagocytosis, crystal cells are

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responsible for melanisation and lamellocytes are required for encapsulation of foreign bodies

(Rizki and Rizki 1984) (Fig. 3B).

The Drosophila hemocytes

Plasmatocytes

Plasmatocytes are the “professional phagocytes”, usually compared to vertebrate

macrophages. With the exception of some glial cells that can function as macrophages in the

developing central nervous system (CNS) (Sonnenfeld and Jacobs 1995), they are the only

cells responsible for the removal of apoptotic cells and microorganisms in embryo, larvae and

adult flies (Wood and Jacinto 2007). One other critical role of plasmatocytes is to produce and

secrete extracellular matrix (ECM) molecules. These are the main source of the ECM

covering all cell surfaces being in contact with the hemolymph. Plasmocytes are highly motile

cells: from stage 10 of embryogenesis, they migrate from their place of origin (head

mesoderm, see below) and disperse into the embryo, following specific migratory routes

(Tepass, Fessler et al. 1994). This migration is possible thanks to very well developed actin

rich filopodia and lamellopodia, that allow not only the moving of plasmatocytes but also

their exploring of the environment (Wood, Faria et al. 2006). One of the main migratory

routes leads along the ventral midline to the CNS, where the plasmatocytes via secretion of

the ECM play a critical role in the proper condensation of the developing nervous system

(Oloffson 2005). This developmentally programmed migration of hemocytes depends on the

expression of the Pvr (receptor) in the plasmatocytes and the Pvf2 and Pvf3 (ligands) by other

embryonic tissues (Cho, Keyes et al. 2002; Wood, Faria et al. 2006). The Pvr is the only

Drosophila homolog of a vertebrate receptor for cytokines of platelet-derived growth factor

(PDGF) family and vascular endothelial growth factor (VEGF) receptor. The fruit fly genome

contains three genes coding for putative Pvr ligands: pvf, pvf1 and pvf3. The Pvr signalling

pathway is also involved in plasmatocyte survival in embryos and plasmatocyte specification

and proliferation in larvae (Munier, Doucet et al. 2002; Bruckner, Kockel et al. 2004; Jung,

Evans et al. 2005).

The plasmatocytes are capable of chemotactic movements toward a tissue wound where

they participate in damage healing. The chemotaxis is however independent of the Pvr

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signalling (Stramer, Wood et al. 2005). The main function of plasmatocytes is phagocytosis.

During development, removal of apoptotic corpses and cellular debris is essential for the

correct placement and shape of newly forming organs. By the end of embryogenesis up to

90% of all plasmatocytes may contain at least one apoptotic body (Tepass, Fessler et al.

1994). The recognition of apoptotic cell requires the function of specific receptors present at

the embryonic phagocyte membrane : Croquemort (CRQ) and Draper (Franc, Dimarcq et al.

1996; Manaka, Kuraishi et al. 2004). The engulfing of invading microorganisms is another

phagocytic function of plasmatocytes that engages several types of receptor proteins, such as

Eater, a EGF-domain phagocytosis receptor expressed exclusively on plasmatocytes. It binds

to and leads to internalisation of a broad range of bacteria (Kocks, Cho et al. 2005). Recently,

Nimrod (NimC1) was described as a receptor present only on larval phagocytes which

increases remarkably the efficiency of bacteria engulfing (Kurucz, Markus et al. 2007). The

scavenger receptor, dSR-CI, is a receptor capable of recognizing both gram-negative and

gram-positive bacteria (Ramet, Pearson et al. 2001). Extremely interesting is the case of the

Dscam proteins (Down syndrome cell adhesion molecule), that belong to the immunoglobulin

(Ig) superfamily. Alternative splicing offers the theoretical possibility of up to 19 000 Dscam

different isoforms. Dscam are expressed inter alia at the hemocyte membrane and take part in

the phagocytosis of bacteria (Watson, Puttmann-Holgado et al. 2005).

Lastly, it was reported that the plasmatocyte mediated phagocytosis could participate

in a kind of “immune memory”, a process so far specific only for vertebrates. Pham and

colleagues have shown that flies primed with a sublethal dose of bacteria (S. pneumoniae) are

protected against a lethal dose of the same bacteria when challenged one week later and this

memory depends on plasmatocytic phagocytosis (Pham 2007).

The phagocytic capacities of plasmatocytes are reinforced by the presence of opsonins

belonging to the family of Thioester-containing proteins (Teps). In the Drosophila genome

there are six different genes coding for Teps. Teps contain several domains shared with the

α2-macroglobulin, and complement (C3) family of proteins, which in vertebrates act as

protease inhibitors and opsonin respectively (Dodds and Law 1998; Lagueux, Perrodou et al.

2000; Blandin and Levashina 2004). It was also suggested that Dscam molecules could bind

directly to bacteria and potentially opsonise them (Watson, Puttmann-Holgado et al. 2005).

Finally, there is another function of plasmatocytes worth to be mentioned as it is

linked to the humoral response. Plasmatocytes have the capacity to secrete the cytokine upd3

that activates JAK/STAT pathway in the fat body, leading to secretion of immune effectors

(Agaisse and Perrimon 2004).

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Fig. 4. The life cycles of Drosophila melanogaster and Leptopilina boulardi. Females of L. boulardii lay eggs in Drosophila larvae. Larvae mount a cellular immune response against parasitoid wasp egg, resulting in encapsulation and melanisation of the egg. The succesful neutralisation of the parasite, permits the development of fly (blue arrows). In case where the Drosophila immune response fails, wasp larvae develop (red arrows) (Photos M.Crozatier).

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Crystal cells

Crystal cells are generated both during embryonic and larval hematopoiesis. Their role

in the embryo remains unknown (Wood and Jacinto 2007), while adult flies are devoid of this

cell type (Lanot, Zachary et al. 2001). In the larval hemolymph, crystal cells make about 5%

of circulating hemocytes They owe their name to the big crystals of zymogen

proPhenylOxydase (proPO) present in their cytoplasm and necessary for the process of

melanisation. Melanisation leads to the deposition of a black pigment that contributes to the

clot at the site of the wound or around foreign bodies in the hemocel (Ashida M. 1997).

Crystal cells function as a storage for large amount of proPO. Once disrupted they release

their content into the hemolymph where the enzymes can function (Meister 2004). ProPO is

cleaved and turned to its active form - phenoloxidase (PO) by the action of a cascade of serine

proteases. PO carries the monophenol mono-oxygenase activity that converts tyrosine to

melanin. Many of intermediate compounds formed during the melanin synthesis are cytotoxic

and could participate into the killing of pathogens (Meister and Lagueux 2003; Meister 2004).

The Drosophila genome contains three genes coding for proPO: doxA1, doxA3 and cg8193,

that are expressed in crystal cells with doxA3 being expressed also in lamellocytes (Irving,

Ubeda et al. 2005). Melanisation at the site of injury in larvae is mediated exclusively by

crystal cells. It is impaired in mutants affecting crystal cell development such as lozenge, or

affecting the release of proPO such as Black cells (Bc) (Rizki T. M. 1980). The source of PO

in the adult where the crystal cell are no longer present is unknown (Lanot, Zachary et al.

2001; Lemaitre and Hoffmann 2007).

Crystal cells do also participate in clot formation. Clotting is critical in limiting

hemolymph loss and creating an immune barrier during wound healing in insects. Clot is a

quickly forming barrier against infection that immobilises bacteria, prevents their spreading

and promotes their killing. The first step of clot formation– preliminary soft clot - is

independent of the crystal cell action, as it can occur even in Bc, or lz mutant larvae.

However, the PO originating from crystal cells acts during clot maturation to produce the hard

clot by cross-linking the preliminary soft clot that tightens the grip of bacteria and thereby

reducing the risk of post-injury infection (Bidla, Lindgren et al. 2005; Lemaitre and

Hoffmann 2007).

Finally, melanisation mediated by crystal cells is engaged in the encapsulation process

that critically requires the third hemocyte type, the lamellocytes.

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Fig. 5. Encapsulation and melanisation of the parasitoid wasp Leptopilina boulardi in

Drosophila larvae

(A) Wasp egg (arrow) in the body cavity of Drosophila melanogaster, soon after deposition; (B) Encapsulated egg. Lamellocytes are clearly observed on the egg surface (arrows); (C) Melanised capsule (arrow) surrounding a wasp egg in a Drosophila larva; (D) Adult fly with two encapsulated wasp eggs (arrows) in its abdomen (after Vass and Nappi 2000 and Carton et al. 2005).

A B

C D

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Lamellocytes

The lamellocytes of Drosophila are flat, large, adhesive cells only produced in the

larval hematopoietic organ – the lymph gland. They are rarely observed in healthy larvae but

differentiate in very large number upon specific challenge like parasitoid wasp egg laying into

the larva. In the nature at least around 50 hymenopterae species are the parasites of

Drosophila (Carton 1986). One species widely used in laboratories to study the Drosophila

immune response is Leptopilina boulardii. Wasp females lay eggs into the hemocel of young

Drosophila larvae and use the host body as an environment for the developing offspring

(Fig.4). The presence of the parasitoid egg triggers a cellular immune response. It is proposed

that the wasp egg is recognised in the larval body by circulating plasmatocytes (Russo, Dupas

et al. 1996). Then the signal of danger is sent to the lymph gland and massive differentiation

of lamellocytes occurs (Crozatier, Ubeda et al. 2004). The lamellocytes are released into the

hemolymph where they stick to the egg and form a multilayered capsule around the invader in

a process called encapsulation (Fig.5). Encapsulation engages all three hemocyte types as the

crystal cells are involved in the melanisation of the capsule. The egg is finally killed, probably

by the effect of cytotoxic free radicals or quinons, although the real reason of its death is not

entirely clear (Vass and Nappi 2000; Meister 2004). Once the egg is encapsulated and

melanised it stays as a scar in the larva and adult (Fig. 5 C,D) but the fly can develop and

hatch normally. If the fly immune response fails, a wasp hatches from the pupa at the expense

of the fly (Fig. 4). Parasitoid wasps use different strategies that can “cheat’ the host response.

In some wasp species like Asobara tabida, the eggs attach easily to the host tissue thanks to a

sticky chorion and finally get embedded in a host site inaccessible to hemocytes (Prevost,

Eslin et al. 2005). Another way to escape is an active suppression based on specific

substances introduced into host by the female wasp at the time of oviposition. For example,

L. heterotoma and L. boulardi inject virus like particles (VLP) produced by the so-called long

glands. The proteins present in the VLP inhibit the encapsulation by changing the morphology

of the lamellocytes, resulting in their diminished adhesive ability (Rizki and Rizki 1984; Rizki

and Rizki 1990; Rizki and Rizki 1990; Rizki, Rizki et al. 1990; Lemaitre and Hoffmann 2007;

Schlenke, Morales et al. 2007). Species devoid of VLP, such as Asobara citri could possibly

directly affect the hematopoietic organ, the lymph gland and induce its degradation (Moreau,

Eslin et al. 2003).

The relationship between Drosophila and parasitoid wasps is an example of the

co-evolution that pushes the species to reciprocal improvement of the physiological

30

prohemocytes

Stage 11

Stage 17

plasmatocytes crystal cells

Stage 5

crystal cellsplasmatocytes

A

srpgcm,

prx

prx

doxA3

doxA3

B prohemocytes

Stage 11

Stage 17

plasmatocytes crystal cells

Stage 5

crystal cellsplasmatocytes

A

srpgcm,

prx

prx

doxA3

doxA3

B prohemocytes

Stage 11

Stage 17

plasmatocytes crystal cells

Stage 5

crystal cellsplasmatocytes

A

srpgcm,

prx

prx

doxA3

doxA3

B

Fig. 6. Embryonic hematopoiesis

(A) Blastoderm fate map of the embryonic hemocytes (EH). The EH anlage is restricted to 70-80% EL (egg length) (red); (mesoderm is in yellow) (after Holz et al. 2003, modified) (B) overlapping of gcm and srp expression in stage 5 embryos, showing the domain of origin of the prohemocytes. (C, E) Plasmatocytes stained with peroxidasin (prx) located in the anterior part of the embryo at stage 11 (C) and dispersed within the whole embryo at stage 17 (E). (D, F) Crystal cells, stained with doxA3 do not migrate but stay localised in the anterior segments in stage 11 (D) and 17 (F). (after Bataille 2006, PhD thesis)

31

capacities. However initiating and maintaining the immune system can be energetically

expensive and have an impact on the fitness of an animal. Parasites and pathogens can

influence the life history of host (Sandland and Minchella 2003). It was reported that

Drosophila melanogaster that successfully defended itself against Asobara tabida exhibited

reduced feeding and thinner puparian walls, but also decreased survival as adults under

conditions of starvation and desiccation (Fellowes A. 1999; Hoang 2001; Tien 2001). The

immunological costs can be masked or revealed depending on environmental conditions.

Additionally, although immunological response might be initiated at a particular

developmental stage, the results can be exhibited at later point in ontogeny (Sandland and

Minchella 2003). Similarly to parasitoidal, bacterial infection can affect the fitness of the fly

in sub-optimal conditions and it was even suggested that the cost of immunity might be

important factor limiting the evolution of resistance in food-limited environments (McKean,

Yourth et al. 2008).

Embryonic hematopoiesis

Embryonic hematopoiesis takes place in the head mesoderm and leads to formation of an

almost invariant number of plasmatocytes (~700) and crystal cells (~30), 95% and 5% of all

hemocytes respectively (Bataille, Auge et al. 2005). The anlage of the embryonic hemocytes

is defined by the expression of the transcription factor Serpent (Srp), a member of the GATA-

binding transcription factors family. Srp is expressed in all hemocyte precursors and is

required for the development of plasmatocytes and crystal cells. Loss of srp leads to the

complete depletion of embryonic hemocytes (EH) (Rehorn, Thelen et al. 1996).

Elegant, homotypic single–cell transplantation experiments done by Holz et al. restricted

the anlage of the EH to a precise region located between 70-80% EL (the posision on the egg

in the anterioposterior axis is given as per cent of Egg Length, where 100% EL is the anterior

tip and 0% the posterior), within the mesoderm corresponding to srp cephalic expression (Fig.

6A,B). They were also able to show that EH are already specified at the blastoderm stage

(Holz, Bossinger et al. 2003).

During embryogenesis, a fraction of hemocytes migrates from its place of birth by

specific paths to seed the whole embryo. These cells are the plasmatocytes, the dedicated

phagocytes. A much smaller fraction of prohemocytes do not migrate and remains localised

32

Fig. 7. The model for the generation of the hematopoietic lineages in the Drosophila embryo

Srp is the earliest expressed gene in the embryonic hemocyte pecursor pool. Most of Srp+ cells begin to express Gcm and differentiate as plasmatocytes. A smaller fraction of Srp+ cells begin to express Lz. ~60% of Lz+ cells maintain the expression of Lz and become crystal cells and the rest will become plasmatocytes. Gcm/Gcm2 and U-shape antagonise the crystal cell fate (after Lebestky et al.. 2000, Bataille et al. 2005, modified).

Srp+

Lz+

Lz+

Gcm+

Gcm2+ Gcm+

Gcm2+

Gcm+

Gcm2+Gcm+

Gcm2+

U-shaped crystal cell

plasmatocyteprohemocyte

Gcm+ , Gcm2+

60%

40%

33

around the proventriculus. These hemocytes are the crystal cells (Fig. 6 C-F) (Tepass, Fessler

et al. 1994; Lebestky, Chang et al. 2000).

The molecular control of embryonic hematopoesis has now been well described.

Several transcription factors and signalling pathways have been reported to be involved in this

process. Two zinc-finger containing transactivators: Gcm ( glial cells missing) and Gcm2 are

required for plasmatocyte development. The crystal cell fate needs the expression of the

transcription factor Lozange ( Lz) (Lebestky, Chang et al. 2000; Waltzer, Ferjoux et al. 2003).

In a detailed work, Bataille et al. showed that Gcm is coexpressed with Srp in all

prohemocytes in early embryos (stage 5) (Fig.6B). Only later (stage 6), the anterior-most cells

of the prohemocyte cluster downregulate gcm thereby allowing the expression of Lz. Only

60% of Lz+ cells will be able to maintain Lz through an autoregulatory loop and acquire a

crystal cell fate, the rest becoming plasmatocytes. In these 40%, it is residual Gcm that

interferes with lz expression and promotes plasmatocyte differentation (Fig.7) (Bataille, Auge

et al. 2005). The fact that gcm, when ectopically expressed, can induce differentiation of all

the prohemocytes into plasmatocytes, (Lebestky, Chang et al. 2000) and that, in absence of

gcm/gcm2, lz can transform all of the hemocytes into crystal cells suggests that embryonic

prohemocytes are bipotent progenitors (Bataille, Auge et al. 2005). Additionally it was shown

that another Drosophila transcription factor, U-shaped (Ush), the homolg of vertebrate Friend

of Gata (FOG), is expressed from stage 8 in plasmatocytes and antagonises crystal cell

development. Figure 7 summarises the molecular control of the embryonic hematopoiesis.

Larval hematopoiesis

Unlike embryonic hematopoiesis, larval hematopoiesis occurs in a specific organ,

called the lymph gland (LG). Although this nomenclature from the literature will be used

here, we have to keep in mind that the insects have no lymph but rather hemolymph, therefore

we should call the hematopoietic fly organ the “hemolymph gland”. Larval hematopoiesis is

more flexible than embryonic hematopoiesis in terms of the total number of cells released and

proportion between plasmatocyte and crystal cell fates. It can also give rise, in specific

conditions, to a third cell type – the lamellocyte.

The lymph gland forms in the embryo, independently of the embryonic prohemocytes.

The LG primordium originates from the cardiogenic mesoderm (as the other Drosophila

34

Fig. 8. Ontogeny of the lymph gland

The earliest known marker of the developing anlage of the larval LG is the gene collier.(A) Expression of col is detected in two discrete clusters of cells in the dorsal mesoderm of the thoracic segments T2 and T3 starting at embryonic stage 11 (germ band extension stage) (black arrows). (B, C) The Col+ clusters migrate toward each other between stage 12 and early 13 and coalesce to form the paired lobes of the lymph gland. (D-F) Expression of col becomes restricted at stage 14 and at stage 16 it marks only a specific posterior region of the LG, the PSC (black arrowheads) (Crozatier et al.. 2004).

(G,H) Expression of the transcription factor Odd indicates that the primordium of the embryonic LG likely comes from three paired clusters of cells located in the thoracic segments T1, T2 and T3. (G-J) Expression of the homeotic gene antp is restricted at 11-12 stage to T3 in the LG (Mandal et al.. 2007).

E F

G

JI

H

Stage 11 Stage 12/13

Stage 14 Stage 16 Col

35

circulatory system cells: cardioblasts and pericardial cells) that is specified within the

mesoderm by signalling pathways, Decapentaplegic (Dpp), Wingless (Wg) and FGF (Cripps

and Olson 2002; Han, Yi et al. 2006). On the blastoderm fate map, it was initially mapped

between 50 and 55% EL in the thoracic lateral mesoderm ((Holz, Bossinger et al. 2003);

however the cells of the LG primordium are not determined to a specific fate before the

second post-blastoderm mitosis. The earliest marker of developing anlagen of the LG is the

gene collier, the expression of which is detected in two discrete clusters of cells in the dorsal

mesoderm of the thoracic segments T2 and T3 starting in embryonic stage 11 (germ band

extension stage). The Col+ clusters migrate toward each other and coalesce to form the paired

lobes of the lymph gland. Afterwards, Collier expression becomes restricted and, at stage 16,

it marks only a specific posterior region of the hematopoietic organ (see below) (Fig. 8 A-F).

Srp is not detected in the LG precursor prior to stage 12. In srp mutant embryo col specific

expression in LG progenitors can be observed, indicating that these cells are specified

independently of srp (Crozatier, Ubeda et al. 2004). Although expression of collier suggests

that the LG origins from two cell clusters, expression of another transcription factor Odd

(Odd-skipped) indicates that the primordium of the embryonic LG more likely comes from

three paired clusters of cells located in the thoracic segments T1, T2 and T3 at stage 15 (Fig.8

G-J) (Mandal, Banerjee et al. 2004). Odd is a zinc-finger protein expressed in segmental

clusters in the dorsal mesoderm of segments T1-A6 (Ward and Skeath 2000). Expression of

the homeotic gene Antennapedia is already restricted at stage 11-12 to the T3 and A1 segment

in the cardiac tissue (Fig. 8 G-J) (Mandal, Martinez-Agosto et al. 2007). Antp colocalizes

with Collier in the posterior region of the LG at stage 14.

The developing LG is localised in the dorsal part of the embryo along the cardiac tube

(Drosophila heart), just posterior to the hormone-secreting organ, the ring gland (analogue of

vertebrate pituitary) (Fig 9A, B). During larval development, the LG grows in size and when

fully developed in third instar larvae, it consist of about 5000 cells per lobe (compared to ~30

at the end of the embryogenesis). In addition to the anterior lobes, which starts to form in the

embryo and become the main Drosophila hematopoietic site, variable numbers of posterior

lobes form during larval development (Fig. 9 C). Although their function is not well

characterised it has been proposed that they constitute a reservoir of prohemocytes. At the

onset of metamorphosis, in response to the ecdysone secretion peak, the lymph gland

disperses and hemocytes are released into circulation (Sorrentino, Carton et al. 2002). It is the

first developmental time when larval hemocytes can be used by Drosophila. Before that, all

circulating hemocytes originate from the embryonic wave of hematopoiesis. The embryonic

36

Fig. 9. Development of the lymph gland (A,B) The lymph gland (LG) is localised in the dorsal part of the embryo in direct contact with the anterior part of the cardiac tube, posterior to the ring gland. (C) During larval stages, the LG grows in size and in the late L3 larva is composed of a pair of anterior lobes, the main hematopoietic site, and several posterior lobes. Circulating embryonic hemocytes (in green) persist throughout embryogenesis and larval development until the adult stage (after Wood and Jacinto 2007). (D) Localisation of the LG in larvae (E) image of the LG in scanning electron microscopy (from Meister and Lagueux 2003).

Larva L3 Lymphgland

A

B C

D E

Lymph gland

37

hemocytes in larval circulation divide actively growing in number from ~200 cells in L1 up to

~2500 in L3 larvae and survive larvae and up to the adult stages (Lanot, Zachary et al.

2001). A majority of larval hemocytes circulates but a fraction, called sessile hemocytes

(containing both plasmatocytes and crystal cells), stays attached to the integument. Therefore,

the hemolymph of the imago contains a mixture of embryonic hemocytes and larval

hemocytes released from the lymph gland. Until now, no haematopoietic site has been

described in the adult fruit fly (Holz, Bossinger et al. 2003).

The detailed work of Jung et al. revealed a very interesting organisation of the

anterior-most lobes of the fully developed L3 LG. Already in the light microscopy one can

observe two different regions: a more rough zone at the periphery of the lobe and a smooth

area inside. The outside zone is called the cortical zone (CZ) and is occupied by differentiated

plasmatocytes and crystal cells. The inner part is called a medullary zone (MZ) and contains

non-differentiated hemocytes (Fig. 10A) Additionally, the Posterior Signalling Center (PSC)

resides in the posterior part of the lobe (Fig.10C) (Lebestky, Jung et al. 2003; Jung, Evans et

al. 2005).

The medullary zone can be visualised by the expression of tep4 and a domeless-gal4

transgene that was isolated from a P-insertion screen for identifying X-linked essential genes

(Fig10 B,C) (Bourbon, Gonzy-Treboul et al. 2002; Irving, Ubeda et al. 2005; Jung, Evans et

al. 2005). Domeless (Dome) is the Drosophila receptor of the JAK/STAT (Janus

Kinase(JAK)/Signal Transducer and Activators of Transcription (STAT) pathway (Fig. 10D).

The fact that Dome is expressed in the MZ is very interesting, knowing that the JAK/STAT

pathway was previously described to be involved in larval hemocyte formation. The

JAK/STAT pathway was first described in vertebrates as the signalling pathway responding to

interferon (Schindler, Fu et al. 1992; Darnell, Kerr et al. 1994). Then in mammals, large

families of cytokines and single-pass transmembrane receptors, named type I cytokine

receptors have been identified (for review (Taga and Kishimoto 1997; O'Shea, Gadina et al.

2002; Heinrich, Behrmann et al. 2003; Kristensen, Kalisz et al. 2005). The JAK/STAT

pathway mediates intra-cellular signalling. JAK kinases are anchored to the intracellular part

of signalling receptors. Binding of the cytokine induces conformational changes in the latter

that bring two JAKs in close proximity. This allows JAK trans-phosphorylation and

phosphorylation of the receptor, thereby creating a docking site for STAT transcription

factors. STATs become in turn phosphorylated, leading to their dimerisation and translocation

into the nucleus where they function as transcriptional regulators. The JAK/STAT signalling

pathway regulates numerous aspects of development and tissue differentiation. Altered

38

Fig. 10. Morphology of the lymph gland.

(A) Nomarski view of the LG in third instar larvae, showing two distinct regions within the primary lobes: a rough cortical zone (CZ) and a smooth medullary zone (MZ) (red arrows). Black arrows indicate the boundary between the CZ and MZ. 1° primary, 2° secondary lobe (from Jung et al.. 2005) (B) The MZ can be visualised by the expression of tep4 (purple). (C) Confocal microscopy view of the LG anterior lobes: the MZ containing prohemocytes is marked with GFP (dome-gal4/UASmCD8GFP) (green), the CZ containing differentiated cells is stained for the proPO (red) and the PSC is stained with anti-Col (blue). (D) Schematic representation of the components of the JAK/STAT signalling pathway in Drosophila (from Arbuzova and Zeidler 2006).

1°lobe

2°lobe

CZ (Cortical zone) differentiatied cells

MZ (Medullary zone)prohemocytes

Col, dome, ProPO

1º

2º

tep4

A

DC

PSC(Posterior Signaling Center)

B1º

2º

39

JAK/STAT activity has been associated with several human diseases including leukaemia,

myocardial hypertrophy and asthma while knock-out studies in mice point to a central role of

JAK/STAT signalling in hematopoeisis and regulation of immune functions (Shuai and Liu

2003; O'Shea and Murray 2008). In contrast to mammals, only one receptor, Domeless

(Dome) (Brown, Hu et al. 2001; Chen, Chen et al. 2002) one JAK (Hopscotch, Hop) (Binari

and Perrimon 1994) and one STAT (Stat 92E or Marelle (Hou, Melnick et al. 1996; Yan,

Small et al. 1996) have been characterised in Drosophila, while three cytokines, Unpaired

(Upd), Upd2 and Upd3 have been shown to act upstream of JAK-STAT signalling (Harrison,

McCoon et al. 1998; Brown, Hu et al. 2001; Chen, Chen et al. 2002; Agaisse, Petersen et al.

2003; Gilbert, Weaver et al. 2005; Hombria, Brown et al. 2005).

Thanks to the conservation during evolution on the one hand and relative simplicity on

the other, Drosophila has become a model of choice for investigating this signalling pathway.

One of the first evidences that JAK/STAT signalling could be involved in Drosophila

hematopoiesis came from analysis of dominant-gain of function mutations of hop. Two

thermosensitive alleles Tum- and T42 were isolated, where the kinase is constitutively active.

hop Tum-l larvae display numerous circulating lamellocytes and melanotic pseudotumors in

absence of wasp infection when kept at restrictive temperature. The effect seems to be LG

autonomous since hop Tum-l LG when transplanted in a wild type host, provoke an

overproliferation of hemocytes and a melanisation phenotype (Hanratty and Dearolf 1993;

Harrison, Binari et al. 1995; Luo, Hanratty et al. 1995; Agaisse and Perrimon 2004).

Conversely, loss of function of hop leads to a complete absence of lamellocyte differentiation

upon wasp infestation. Again, the phenotype seems to be related to hop function in the LG

since no effect on the number on circulating cells was observed (Sorrentino, Melk et al.

2004). Aside, while one of the JAK/STAT ligands, upd3, was previously reported to be

secreted by circulating plasmatocytes upon septic injury, Jung et al. have shown that an

upd3gal4 construct drives the expression of the reporter gene in the MZ and in the PSC.

Together these data suggested that the JAK/STAT pathway could be active in the LG in

controlling hematopoiesis (Jung, Evans et al. 2005). Function of tep4, another gene

specifically expressed in the MZ, remains unknown (Irving, Ubeda et al. 2005).

The cortical zone (CZ) is occupied by differentiated hemocytes and is defined mainly by

expression of markers of mature hemocytes and lack of expression of MZ markers (Fig. 10C).

By elegant cell tracing experiments, Jung et al have shown that the cortical zone cells

originate from the medullary zone. In second instar larvae, all hemocytes are in a precursor

40

Fig. 11a. col is required for the formation of the PSC

(A,C,E) wild type, (B,D,F) col1 LG. The col1 mutant phenotype is the loss of the PSC.

Expression of PSC markers, Ser-lacZ and Ser is lost (arrowhead in C,E) while the expression

of Ser in scattered cells (arrows E,F) remains unchanged. Bar:50μm (from Crozatier et al.

2004).

Fig. 11b. PSC plays an instructive role in lamellocyte production

(A,C,E) wild type, (B, D, F) col1 LG. (A,B) col1 lymph glands show increased numbers of

crystal cells (stained with doxA3) in comparison to wt. (E,F) When challenged by parasitoid

wasps, col1 larvae are unable to produce lamellocytes (stained with specific antibody against

an integrin αPS4), while wt larvae lymph glands are full of newly differentatied lamellocytes

Bar: 50μm (from Crozatier et al. 2004).

41

state and reside in the MZ. CZ forms with the progressive downregulation of domeless and

differentiation of hemocytes in third instar larvae (Jung, Evans et al. 2005). The exact mode

of the transition from the medullary zone toward the cortical zone remains a challenging

question. The cell lineage of larval hemocytes has not been documented up to now and still

needs to be resolved.

The Posterior Signalling Centre (PSC) was described for the first time by Lebetzky and

colleagues in 2003, when they observed that Serrate (Ser), a Notch (N) ligand was expressed

in a small population of lymph gland cells, localised in the posterior part of the anterior lobe.

This is where the name “PSC” comes from. Ser expression could be seen with the use of an

antibody or expression of Ser-lacZ reporter gene. In addition to the PSC, the antibody

revealed the expression of Ser in scattered cells throughout the gland (Fig. 11a, C-F),

(Lebestky, Jung et al. 2003). Notch signalling is an evolutionary conserved signalling

pathway that regulates cell fate decisions in numerous developmental systems and involves

direct cell-cell interaction (for review see (Bolos, Grego-Bessa et al. 2007; Carlson, O'Connor

et al. 2007; Fiuza and Arias 2007; Nichols, Miyamoto et al. 2007). Several reports have

documented the involvement of N signalling in crystal cell formation. In Nts larvae (Nts is a

termosensitive allele of Notch receptor), raised at restrictive temperature (29ºC) no crystal cell

forms (Duvic, Hoffmann et al. 2002). Based on this mutant phenotype and Ser expression in

the late LG, Lebetzky et al. proposed that Ser+ cells migrate from the PSC and seed the lobe

where they instruct the neighbouring cells to become crystal cells. The proposition that the

PSC controls proper larval hematopoiesis was also based on the observation that Ser and not

Delta (the other Drosophila Notch ligand), is necessary for crystal cell formation.

Unfortunately, not all of the PSC hypothesis turned to be right. In 2004, Crozatier and

colleagues showed that collier which codes for a transcription factor, orthologous to

vertebrate Early B-cell Factor (EBF) is expressed specifically in the PSC of the lymph gland.

One of the phenotypes displayed by col1 mutant larvae (col1 is a null allele of collier), is the

loss of the PSC. While, as a consequence, Ser expression is lost from the PSC, it remains

unchanged in scattered cells in the anterior lobes of col1 mutant LG (Fig. 11a). Likewise,

crystal cells form correctly (Fig. 11b) (Crozatier, Ubeda et al. 2004). The differentiation of

crystal cells in col1 therefore showed that it is Ser activity in the scattered cells and not in the

PSC that is necessary for crystal cell formation. However Lebetzky’s idea of a signalling

center was correct, as it turned out that the PSC controls other aspects of larval hematopoiesis.

The loss of the PSC in col1 has indeed dramatic consequences in terms of hemocyte formation

42

S1PSCCol

lamellocytesL

S2

pro-hemocyte

wasp egg

circulatingplasmatocytes

L

Fig. 12. A model for the induction of lamellocyte differentiation in response to wasp parasitation. Col enables the PSC cells to respond to a primary signal (S1), that is likely emitted by plasmatocytes after their contact with the wasp egg. As a result, the PSC sends a secondary signal (S2) to prohemocytes, inducing their massive differentation into lamellocytes (after Crozatier et al. 2004, modified).

43

(see below). The first observation was that PSC-depleted larvae are unable to mount a

cellular immune response, i.e. there is no differentiation of lamellocytes in response to egg-

laying by the parasitoid wasp Leptopilina boulardii (Fig.11b). In wild type larvae, col-

expressing cells remain restricted to the PSC upon wasp infestation, while lamellocytes

differentiate from the MZ, showing that PSC cells are not the precursors of lamellocytes but

rather that they play an instructive role in orienting prohemocytes to differentiate into

lamellocytes. The following model of cellular response upon infestation was proposed: Col

enables PSC cells to respond to a primary signal (S1) likely emitted by plasmatocytes upon

their encounter of a parasitoid egg. Upon receiving S1, PSC cells send a secondary signal (S2)

that orients prohemocytes to develop into lamellocytes (Fig. 12) (Crozatier, Ubeda et al.

2004).

Later work showed that the Posterior Signalling Center can first be identified in the

embryo by the expression of Antennapedia in stage 11-12 when the primordium of the lymph

gland starts to form. The homeodomain cofactor Homothorax (Hth) is initially expressed

ubiquitously in the embryonic LG but is soon downregulated in the Antp+ cells. The mutually

exclusive expression pattern of Antp and Hth specifices two LG areas: Antp specifies the PSC

cells and Hth, the rest of the gland (Mandal, Martinez-Agosto et al. 2007). In parallel, collier

expression starts to be restricted to the PSC around stage 14 where it persists up to the end of

L3 (see above) (Crozatier, Ubeda et al. 2004). Then Serrate starts to be expressed in the PSC

in second instar larvae (Lebetzky) as does hedgehog (Fig. 13), while downstream components

of the Hh signalling pathway, the receptor Patched (Ptc) and transcription factor Cubitus

interruptus (Ci) are expressed in the medullary zone. Together, these led to the proposal that

Hh produced in the PSC diffuses and signals through activated Ci in the medullary zone,

keeping the MZ cells in a precursor state (Mandal, Martinez-Agosto et al. 2007).

Control of larval hematopoiesis

Contrary to embryonic hematopoiesis, the control of Drosophila larval hematopoiesis has

not been characterised in details. In 2004, when I started my PhD work, the information in the

literature was still very fragmentary and there was not a clear image of overall regulation of

hemocyte formation in the lymph gland. Only few links were made between the embryonic

and larval waves of hematopoiesis. One of them was the transcription factor Lozenge (Lz)

44

Fig.13. Expression of PSC markers during LG development (A) The Posterior Signalling Center can first be identified in the embryo by the expression of Antennapedia (Antp) in stage 11-12 when the primordium of the lymph gland starts to form. The homeodomain co-factor Homothorax (Hth) initially expressed ubiquitously in the embryonic LG is downregulated in the Antp+ cells. (B) Collier expression starts to be restricted to the PSC at around stage 14 where it persists up to the end of L3. (C,D) Serrate (Ser) and Hedgehog (Hh) start to be expressed in second instar larvae.

45

that is required for crystal cell specification and seems to be common to both embryonic and

larval phases of hematopoiesis, since lz mutant embryos and larvae lack this cell type

(Lebestky, Chang et al. 2000). Otherwise, for example Notch signalling that has been shown

to be necessary for crystal cell formation in larvae, is apparently not required during the first

hematopoietic wave (Duvic, Hoffmann et al. 2002; Bataille, Auge et al. 2005). It should be

noted, however, that even though we know that N controls the larval crystal cell fate, the

temporal window of this requirement has not been defined. The embryonic plasmatocyte fate

specific factors Gcm and Gcm2 are not expressed in the lymph gland (Bataille, Auge et al.

2005). The only information about requirement for larval plasmatocyte specification comes

from the studies of Jung and colleagues showing that Pvr signalling specifically controls the

differentiation of phagocytes (Jung, Evans et al. 2005). Support for the involvement of

JAK/STAT signalling in larval hematopoiesis has already been presented above. Interestingly

a phenotype similar to gain-of-function phenotype of hop (hop Tum-l), that is a massive

differentiation of lamellocytes in absence of immune challenge, was described for

hyperactivation of the Toll pathway. This cellular phenotype appears to be independent from

Toll function in the humoral immune response in the fat body (Crozatier M. personal

communication, (Qiu, Pan et al. 1998). A more precise view of control of larval

hematopoiesis has started to emerge with the characterisation of the PSC.

Collier, a transcription factor with multiple developmental functions

Crozatier et al. in 2004 showed that the transcription factor Collier (Col) is crucial for the

proper function of the PSC. The gene coding for this protein was first characterised by the

group of A. Vincent in 1996 and since then, it has been at the center of interest of the team

(Crozatier, Valle et al. 1996). Col is a founding member of the family of COE transcription

factors, COE for Collier/Olfactory-1/Early B cell Factor isolated from Drosophila, rat and

mouse respectively (Hagman, Belanger et al. 1993; Wang and Reed 1993; Crozatier, Valle et

al. 1996). While there is no evidence for coe genes either in protozoa, fungi or plants,

identification of a coe gene in cnidarians and sponges suggests that the coe genes appeared

with Metazoa (Pang, Matus et al. 2004). While up to four ebf paralogs have been identified in

vertebrates, a single coe member has been identified in all other animals for which genome

sequence is available (NCBI, mars 2008), indicating that expansion of the coe gene family

46

Fig. 14. Schematic representation and alignment of mouse EBF/COE1 and Drosophila Col. The regions corresponding to the DNA Binding Domain (DBD) and the IPT domain are shown in red and yellow, respectively. The ancestral Helix-Loop-Helix (HLH) motif is represented by two separate black boxes (helices H1 and H2a) and the H1-H2 linker in green. The duplicated helix (H2d) specific to the vertebrate proteins is indicated by a blue box and the C-terminal transactivation domain is in grey. EBF and Col show very significant sequence identity; 86% in the DBD domain and 89% in the IPT domain. (from Daburon et al. 2008).

86% 89% Sequence identity %

47

occurred at the origin of vertebrates (Garel, Marin et al. 1997; Wang, Tsai et al. 1997; Wang,

Betz et al. 2002; Daburon, Mella et al. 2008). Biochemical dissection of mouse EBF

identified three functional domains: i) an amino-terminal DNA binding domain (DBD), that

became the signature of the COE family of transcription factors, ii) a Helix-Loop-Helix

(HLH) dimerisation motif showing limited sequence similarity with HLH motifs described in

basic helix-loop-helix proteins (b-HLH), iii) a transcription activating domain. An Ig-

like/Plexin/Transcription factor (IPT) domain with unknown function is also present between

the DBD and HLH domains (Bork, Doerks et al. 1999). Comparison between Col and EBF

showed that the DBD, IPT and HLH domains have been particularly well conserved during

evolution with one noticeable exception that in vertebrates one of the α-helix of the HLH, has

been duplicated (Fig 14) (Dubois and Vincent 2001; Daburon, Mella et al. 2008). EBF and

Col bind to DNA as homodimers and homodimer formation is mediated by the HLH domain

(Daburon, Mella et al. 2008).

Collier was first described as a regulator of head patterning in Drosophila (Crozatier,

Valle et al. 1996); ebf(1) was first characterised as a crucial factor for B-lymphocyte

specification and differentiation (Travis, Hagman et al. 1993; Rothenberg 2000) and olf-1 was

isolated as a gene coding for an olfactory neuron specific transcription factor (Kudrycki,

Stein-Izsak et al. 1993). Further studies of the function of col and ebfs showed that these

genes are involved in the development of several different tissues. In Drosophila in addition

to the head, Col is expressed and required in a specific somatic muscle (muscle DA3), the

wing, and subsets of neurons in the central and peripheral nervous system. (Crozatier, Valle et

al. 1996; Crozatier, Valle et al. 1999; Crozatier and Vincent 1999; Vervoort, Crozatier et al.

1999; Dubois, Enriquez et al. 2007; Crozatier and Vincent 2008). Finally it is also required to

specify the PSC (see above) in the lymph gland (Crozatier, Ubeda et al. 2004). During mouse

embryogenesis, ebf genes are expressed in the limb buds, immature olfactory neuronal

precursors, mature neurons of adult olfactory epithelium, developing nervous system (ebf1-3)

(Garel, Marin et al. 1997; Wang, Tsai et al. 1997; Mella, Soula et al. 2004), progenitors of

facial branchiomotor neurons (fbn) (ebf2,3), mature fbn (ebf1), the genital bulbs and the

harderian gland (ebf4); ebf3 and 4 were detected in human placenta (Garel, Garcia-

Dominguez et al. 2000; Pattyn, Hirsch et al. 2000; Mella, Soula et al. 2004; Asai, Yamaki et

al. 2008). Of particular interest in the context of my studies, is the recent finding that mouse

ebf2 is expressed in immature osteoblasts that constitute a major component of the HSC niche

(Kieslinger, Folberth et al. 2005; Wilson and Trumpp 2006). This expression of ebf2 in cells

controlling the hematopoiesis in bone marrow in the vertebrates raised the fascinating

48

Endostealniche

vascularniche

A

B

C

D

sinusoids

Endostealniche

vascularniche

A

B

C

D

sinusoids

Fig. 15. HSC niches in vertebrates

(A-C) Schematic organisation of a long bone and (C-D) localisation of stem cell niches. The endosteal niche, of which the main components are the osteoblasts is located in the inner surface of the bone at the interface between the bone and bone marrow. The vascular niche is made by endothelial cells of blood vessels – sinusoids. (D) Both niches express regulatory components that influence stem cell functions (after Shiozawa et al..2008, Kiel and Morrison 2008).

49

possibility of a functional analogy between the roles of Col and EBF2 in Drosophila and

vertebrate hematopoiesis, respectively.

Osteoblasts, hematopoietic stem cells and their niche

Osteobasts are the bone forming cells, which secrete bone extracellular matrix during

ossification. They reside in the endosteum, the inner surface of the bone at the interface

between the bone and bone marrow. Osteoblasts line this surface to regulate bone remodelling

and hematopoiesis. Osteblasts are a subpopulation of bone marrow stromal cells, the non-

hematopoietic cells present in hematopoietic tissue that are proposed to secrete factors

regulating hematopoiesis and stem cell functions. The other main cell types in this group are

osteoclasts, endothelial cells, fibroblasts, reticular cells and adipocytes (Fig. 15) (Taichman

2005; Wilson and Trumpp 2006; Kiel and Morrison 2008; Shiozawa, Havens et al. 2008) The

bone marrow of vertebrates is the main adult hematopoietic site containing the hematopoietic

stem cells (HSC).

Hematopoiesis in vertebrates can be seen as a pyramidal process with the cell having

the largest potential at the top (primitive HSC) and terminally differentiated at the bottom

(Fig. 16). Vertebrate blood cells can be classified into two major groups: myeloid and

lymphoid, which have a common precursor. Myeloid cells include erythrocytes,

megakaryocytic, granulocytes and macrophages, whereas the lymphoid precursors give rise to

lymphocytes and natural-killers (NK) cells (Fig. 16) (Shiozawa, Havens et al. 2008)

Mature murine hematopoietic stem cells are described as multipotential progenitors with

the capacity to provide life-long reconstitution of all blood cell lineages (both myeloid and

lymphoid) after transplantation into lethally irradiated recipients (Domen and Weissman

1999). In adults, marrow-derived HSC are on one hand the main source of all mature blood

cells and on the other hand retain the capacity to self-renew (Osawa, Hanada et al. 1996; Watt

and Hogan 2000; Fuchs, Tumbar et al. 2004). The self-renewing of HSC and the balance

between quiescence and differentiation were proposed in 1978 by Shofield et al. to depend on

the specific inductive microenvironment called a stem cell niche (Schofield 1978). Since

then, the interest in the environmental influence on stem cell behaviour in hematopoietic and

other tissue system has kept increasing. Currently we know that there are at least two

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Fig. 16. Blood cell lineages in vertebrates Vertebrate blood cells can be classified into two major classes: myeloid and lymphoid

cells. Myeolid cells include erythrocytes, megacaryocytes, granulocytes and macrophages, whereas the lymphoid precursors give the lymphocytes and natural-killer (NK) cells. Hematopoiesis can be seen as a pyramidal process with HSC having the biggest potential at the top and differentiated cells at the bottom (after Lensch W. http://daley.med.harvard.edu/assets/Willy/Willy_noframes.htm modified).

Myeloid line Lymphoid line

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components of the HSC niche in the bone marrow: the endosteal and vascular niches. (Fig.

15)

The vascular cells have a crucial role in the development and expansion of embryonic

HSC but also in regulation of adult stem cells. During metazoan evolution, the use of bones as

hematopoietic site seems to have appeared with amphibians (Zon 1995; Glomski, Tamburlin

et al. 1997). Up to then, blood cell formation occurred in association with the dorsal vessel, in

the kidney and thymus, as it takes place for example in zebrafish (Murayama, Kissa et al.

2006).

The endosteum is strongly vascularised (De Bruyn, Breen et al. 1970) and endothelial

cells can promote the maintenance of the HSC in culture (Li, Johnson et al. 2004). In the bone

marrow, a large proportion of HSC is attached to the fenestrated endothelium of the blood

vessels called sinusoids. Sinusoids are present in hematopoietic tissues. They have thin walls

formed by a discontinuous, irregularly shaped endothelium that allows cells to pass in and out

of circulation (Kiel, Yilmaz et al. 2005; Wilson and Trumpp 2006). The location of HSCs in

proximity of sinusoids might enable them to constantly monitor the concentration of blood

carried factors that themselves reflect the status of the blood system. Under hematopoetic

stress, a rapid and robust response could be mounted and if necessary more HSCs could be

recruited (Wilson and Trumpp 2006). The endothelial cells of sinusoids, which were shown to

secrete cytokines and express adhesion molecules constitute the vascular HSC niche (Fig.

15) (Rafii, Mohle et al. 1997; Avecilla, Hattori et al. 2004; Kiel, Yilmaz et al. 2005).

Beside the vascular niche, many HSCs within the bone marrow reside at, or near the

endosteum. Endosteal cells secrete factors that promote HSC maintenance and constitute the

stem cells microenvironment, called endosteal niche (Li and Xie 2005; Suda, Arai et al.

2005). A few years ago, osteblasts were recognised as a crucial component of the endosteal

HSC niche in mouse. It was shown that the number of osteoblasts regulates the niche size and

influences the number of HSC (Calvi, Adams et al. 2003; Zhang, Niu et al. 2003). Although

they are not the only source of these factors, osteoblasts were suggested to secrete regulators

of HSC properties, like angiopoietin (Arai, Hirao et al. 2004) and trombopoietin (Qian, Buza-

Vidas et al. 2007; Yoshihara, Arai et al. 2007) or regulators of HSC migration and localisation

into bone marrow, like CXC – chemokine ligand 12 (CXCL12) (Peled, Petit et al. 1999; Petit,

Szyper-Kravitz et al. 2002). A question that still remains to be clarified is how HSC can

access to these factors. Is a direct contact with osteoblasts necessary or could the reception of

diffusible factors occur without the physical contact with the niche? (Kiel and Morrison

2008).

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Fig. 17 A model for the endosteal niche; the stem cell synapse. Schematic representation of the niche-stem cell synapse showing the complex

communication between these two cell populations. The diagram shows putative ligand-receptor interactions and adhesion molecules, as well as some of the intracellular pathways that are activated following signalling (from Wilson and Trumpp 2006).

53

The idea of a physical contact was favoured because of the observation that N-

cadherin homotypic adhesion occurs between the osteoblasts and HSC (Zhang, Niu et al.

2003). The other indication in support of this hypothesis was that osteoblasts promote HSC

maintenance by expressing the Notch ligand Jagged1, thereby activating Notch present on

HSC (Calvi, Adams et al. 2003). Although both interactions could play very important roles

in HSC-niche communication, neither N-cadherin depletion from adult HSC nor conditional

deletion of Notch 1 and Jagged1 from bone marrow does, however, affect HSC maintenance

in vivo (Mancini, Mantei et al. 2005; Kiel, Radice et al. 2007). Numerous other signalling

pathways were proposed to contribute the niche-HSC interaction. A model for a so called

endosteal niche- stem-cell synapse was proposed which postulates the existence of a

dedicated zone for molecular crosstalk between cadherins, integrins, chemokines, cytokines,

signalling molecules and their receptors (Fig. 17) (Wilson and Trumpp 2006). In addition to

the overall complexity of the endosteal niche, it is probable that the endosteal and vascular

niches cooperate in order to control HSC (Fig. 15) (Wilson and Trumpp 2006); (Sugiyama,

Kohara et al. 2006).

Finally, it was also proposed that HSC have the ability to sense ambient calcium

thanks to the expression of the calcium receptor (CaR), and this ability was recognised as

crucial to HSC homing and retention in the bone marrow. Bone is an unique organ with a

much higher Ca2+ concentration then found in the serum, especially near osteoclasts which

resorbe bone. Fluctuations of Ca2+ concentration in different physiological conditions could

influence the activity of HSCs (Adams, Chabner et al. 2006; Porter and Calvi 2008).

As one can see, 30 years after the term niche was first coined, our knowledge about

how the microenvironment influences the stem cell character of HSC in vertebrates is still

fragmentary and often contradictory.

The hematopoietic stem cells

Our knowledge of the hematopoietic stem cell themselves is also incomplete. These

cells are very rare, ~0.05% of total cell number in the bone marrow (Morrison, Uchida et al.

1995). They are not easily accessible as they home to the inside bone surface. Neither a single

specific marker, nor a biological property if employed alone allows for the isolation of a pure

HSC population and a combination of several markers is used to define the HSC. The most

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relevant markers among others are: surface antigen / receptors (Sca-1+, c-Kit+, CD34-), lack

for lineage specification markers ( Lin-), low staining with metabolic fluorochromes such as

DNA marker Ho3342, small size, quiescence (only ~1-3% of HSC are engaged in a cell cycle

(Goodell, Brose et al. 1996); reviewed in (Ratajczak 2008). The difficulty can be even higher

when taking under consideration that the characteristics of HSC vary with age of an animal,

cell cycle status or the source from where these cells were isolated (embryo or adult tissue),

(Ogawa 2002; Quesenberry, Dooner et al. 2005). Indeed, adult HSC have a long

developmental history before they home the bone marrow. During this long developmental

journey, changes in antigen expression on HSCs are observed (reviewed in (Ratajczak 2008).

During mouse embryogenesis, hematopoiesis occurs at several sites. The extra-embryonic

yolk sac and intra-embryonic aorta-gonad –mesonephros (AGM) region both generate HSCs.

Later, secondary sites of hematopoiesis are the liver, spleen, thymus and finally the bone

marrow which doe not generate new stem cells but are thought to be colonised by cells

generated in the primary sites. The fetal liver constitutes the main place of expansion of

HSCs; here HSCs expand in number and acquire the final characteristic surface markers

which define them in the bone marrow (reviewed in (Dzierzak 2002).

The functional test for the HSC character requires HSC to be able to reconstitute the

hematopoietic system of lethally irradiated recipients for long term (Domen and Weissman

1999). In 2004, Matsuzaki et al. have shown that a single bona fide HSC is able to give rise to

all hematopoietic lineages and restore long–term hematopoiesis. That result led to the

suggestion that the high marrow-seeding efficiency is a specific characteristic of primitive

HSCs (the pluriotent HSC with the highest potency) in addition to self-renewal and

multipotent capacity (Matsuzaki, Kinjo et al. 2004).

There are so many data about the vertebrate HSCs and their niche in the literature, that

a synthetic view is becoming virtually impossible. Fortunately, last years have brought a very

convenient model system to study the communication between the stem cells and their niche,

that is the communication between the male and female germline stem cell (GSCs) and their

respective niches in Drosophila melanogaster. The easy accessibility, one-cell resolution and

possibility of detailed genetic analysis favours a transition of results and concepts from

relative simplicity of invertebrate models to complexity of vertebrate mechanisms.

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Fig. 18. Female and male germline stem cells in their gonadal niches. (A) Schematic representation of the organisation of Drosophila ovariole. Cap cells

constitute the niche for the GSC female germline stem cells (GSC, blue), which give rise to cytoblasts (CB, light blue). The somatic stem cells (SSC) give raise to somatic cells (SC), and escorting stem cells (ESC) give raise to escorting cells (EC) spectrosome (S). (B) Female GSCs, SSCc and ESCs are maintained in by the niche through various signalling pathways. Dpp signalling plays the crucial role in GSC maintenance. (C) Schematic represantation of the organisation of the testis. Hub cells are the niche of GSCs and SSCs. (D) GSCs survival is controlled by the hub, mainly through JAK/STAT signalling. In both sexes the GSC are anchored to the niche by ahderent junctions. When a GSC divides the daughter cell that stays in contact with the niche retains the stem cell character, while the one that is displaced away, receives the weaker signal from the niche and starts to differentiate giving raise to gonialblasts (GB) (light blue) (A,C after Fuller and Spradling 2007, modified; B, D after Harrison and Harrison 2006, modified)

A. B.

SSC

ESCEC

SSC SCS

Cap cells

SC

SC

CAPCad/ß-cat

SSC

ECESC

CBGSCDpp,Hh

JAK/STAT

Dpp, Hh, WgJAK/STAT

HUBCad/ß-cat

GBGSC

SCSSC

JAK/STATDpp

JAK/STAT

C. D.

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Drosophila melanogaster stem cell niches as a model for vertebrate systems

Stem cells are defined by their ability to both self-renew and differentiate into mature

cell types, a characteristic that is usually controlled by the stem cell niche. The balance

between self-renewal and differentiation is achieved through an interplay of programs

intrinsic to the stem cells and extrinsic factors sent from the niche. In Drosophila, the neural

and gonadal stem sells were characterised in details and it appears that these two types of cells

use contrasting mechanisms to control their self-renewal. In neural stem cells, self-renewal is

based primarily on asymmetric segregation of intrinsic factors. Specific cellular proteins are

asymmetrically localised in the dividing stem cells, such that only one daughter cell inherits

the determination factors and undergoes differentiation, whereas the other one maintains the

stem cell character (reviewed in (Wodarz 2005; Chia, Somers et al. 2008). Regulation of self-

renewal in germline stem cells is mainly controlled by extrinsic factors, coming from the

niche, although some intrinsic factors are involved as well (Harrison and Harrison 2006).

Drosophila germline stem cells (GSCs) are probably the best characterised adult stem

cells in any organism (for review (Lin 2002; Li and Xie 2005; Harrison and Harrison 2006;

Fuller and Spradling 2007). Whereas the detailed description of the oogenesis and

spermatogenesis is beyond the scope of this introduction, I will mention the discovery of

signalling pathways and mechanisms which constitute the communication between GSC and

their niches.

The ovaries and testis contain distinct stem cell populations for germline stem cells

and somatic stem cells. The external cues are supplied by the microenvironment, the niche,

localised at the tip of the gonad and produced by specialised cells called cap cells in the ovary

(precisely in the ovarioles that are 12-16 functional units in each of two fly ovaries) and hub

in the testis. The male and female niches accommodate 6-12 and 2-3 GSCs, respectively (Fig.

18) (Fuller and Spradling 2007).

In the female ovariole, each GSC divides asymmetrically such that one daughter cell

gets displaced away from the niche and allowed to differentiate. This cell is called a

cystoblast and will further divide to give the egg. In addition to GSCs, there are two

populations of somatic derived stem cells: escorting stem cells (ESC) located near the cap and

giving rise to escort cells and somatic stem cells located more posteriorly (Fig. 18) (Harrison

and Harrison 2006).

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The male niche, the hub, is in direct contact with both germline stem cells and a single

population of somatic stem cells. Like in ovarioles, GSCs divide asymmetrically, which

pushes one cell out of the niche. The non-stem cell daughter is called a gonialblast and will

give rise to spermatogonia (Fig. 18) (Harrison and Harrison 2006).

In both sexes, GSCs are maintained in contact with the niche cells via adherent

junctions rich in E-cadherin and β-catenin, (Song, Zhu et al. 2002; Yamashita, Jones et al.

2003). Disrupting these junction causes stem cell loss, showing that they are both required for

holding GSC in the niche and keeping them away from differentiation factors (Fig. 18B, D)

(Gonzalez-Reyes 2003).

In the GSCs- niche interaction, it is very important to secure the asymmetric division,

that allows both self renewal and differentiation. In a recent work, Yamashita et al. have

proposed that in the testis, the centrosome that localises near the cortex where it attaches to

the hub, controls the spindle orientation leading to asymmetric division (Yamashita,

Mahowald et al. 2007). Asymmetric division of GSCs in ovarioles is controlled by the

spectrosome, a structure comprised of cytoskeletal and regulatory proteins. The spectrosome

is associated with one pole of the mitotic spindle and orients the axis of cell division, resulting

in asymmetric positioning of daughter cells, one staying in contact with the cap (niche) and

the other being pushed away (Lin, Yue et al. 1994; Deng and Lin 1997; Lin and Spradling

1997).

The numerous studies that were performed on the communication between Drosophila

GSCs and their respective niches have revealed the involvement of several signalling

pathways. Maintenance of female GSCs strictly requires the Dpp pathway (homolog of

vertebrate bone morphogenetic protein BMP), mediated by two ligands, decapentapleplegic

(Dpp) and glass-bottomed boat (Gbb) expressed by cap cells (Xie and Spradling 1998; Xie

and Spradling 2000). Dpp signalling represses expression of Bam (Bag of marbles), a key

differentiation regulator. When GSCs divide the repression mediated by Dpp is relieved in the

cell that looses contact with the niche (Chen and McKearin 2003). Additionally Hedgehog

(Hh) and JAK/STAT signalling have proposed to be involved in the maintaining of female

GSCs. (Cox, Chao et al. 1998; King and Lin 1999; King, Szakmary et al. 2001; Lopez-

Onieva, Fernandez-Minan et al. 2008; Wang, Li et al. 2008). Cap cells also secrete Hh, Upd

and Wg (Wingless) that are required for maintenance and proliferation of SSCc. This could be

an interesting example of paracrine signalling since, unlike GSCs, SSCc are not all in direct

contact with the cap (Zhang and Kalderon 2001; Song and Xie 2003). ESCs, which share the

anterior niche with GSCs also depend upon JAK/STAT signalling for their maintenance and

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the loss of JAK/STAT signalling specifically in the ESCs causes their loss (Decotto and

Spradling 2005).

The male GSCs are also maintained by hub cells through Dpp signalling; however the

primary task of controlling the GSC character is performed by the JAK/STAT signalling. The

hub secretes the cytokine-like ligand Unpaired that activates the pathway in the GSC to

induce self-renewal (Kiger, Jones et al. 2001; Tulina and Matunis 2001). When a male GSC

divides, the daughter cell that stays in contact with niche retain the stem cell character, while

the one that is displaced away, receives the weaker signal from hub and starts to differentiate.

The decision of differentiation can be reinforced by the signal relayed to this gonioblast from

somatic supporting cells (Tulina and Matunis 2001). The GSCs and SSCc share the niche, the

hub, that expresses Upd and both require JAK/STAT signalling for their stemness (Fig. 18, B,

(Harrison and Harrison 2006).

As one can see in both the female and male niches, although some signals are different

in these two models, the balance between self-renewal and differentiation is maintained

through asymmetric division resulting in limited access of one daughter cell to a local signal

coming from the niche.

The information taken from the Drosophila GSCs is extremely precious when

analysing the influence of the microenvironment on the behaviour of stem cells in other

tissues or organisms. Comparing the vertebrate stem cells with the fly stem cells can bring

new ideas about the mechanisms and signalling pathways acting in the higher organisms.

JAK/STAT, BMP, Shh (in Drosophila Hh) and Wnt (in Drosophila Wingless) that are critical

in fly GSCs were reported to operate in vertebrate HSC and their niche in the bone marrow,

suggesting the conservation of “stemness” and ‘nichness’ in phyla even early separated in the

evolution (Fig. 17) (Wilson and Trumpp 2006).

The GSCs are not the only Drosophila model of stem cells. Recent reports have

described the existence of different types of adult stem cells in the fly, including neural,

intestinal and Malphigian tubes stem cells (Doe, Fuerstenberg et al. 1998; Micchelli and

Perrimon 2006; Ohlstein and Spradling 2006). Paradoxally however, description of model of

hematopoietic stem cell niche is still missing.

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Hematopoietic stem cells in Drosophila?

During my PhD work, I have studied the hematopoiesis taking place in the Drosophila

larval hematopoietic organ, the lymph gland. Analysis of the organisation and physiology of

this tissue revealed many intriguing similarities between the formation of Drosophila

hemocytes and vertebrate immune blood cells. The most fascinating is perhaps the role of the

Posterior Signalling Center, which can be defined as a group of cells expressing Collier, the

Drosophila ortholog of EBF. The objective of my work was to characterise, at the cellular and

molecular levels, the control of larval hematopoiesis and the communication between the PSC

and hemocyte precursors residing in the medullary zone of the lymph gland. One immediate

question, which I addressed, was: could the PSC act as microenvironmental niche? and if yes,

are there stem cells among the precursors? Another question was that of the cell lineages in

the lymph gland: how and when are cell fate decisions, such as priming to differentiate into

either a plasmatocyte or a crystal cell, taken? What happens when responding to parasitoid

wasp attack demands the massive differentiation of lamellocytes, a cell type that is absent in

healthy larvae? What is the level of lineage plasticity?

As one can see, there were many questions. I will describe in next sections my attempt

to answer some of them and then discuss the new questions, which arose from my work.

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Materials, methods and experimental

design

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67

The materials and methods corresponding to the part of my PhD work published in the

article ”Control of blood cell homeostasis in Drosophila larvae by the posterior signalling

centre."(Krzemien, Dubois et al. 2007), are described in the article and therefore not repeated

here. I rather detail below other experimental procedures, particularly those that I used in the

work done over the last year.

Antibodies and reagents.

The following primary antibodies were used at the dilution indicated in parentheses. Mouse

monoclonal antibodies : anti-Fibrillarin 72B9 (1:1) (gift from J. Cavaillé Toulouse, (Reimer,

Pollard et al. 1987); anti-CycE (1:15) (gift from B. Bello and H. Richardson); anti-DE-

cadherin (1:1); anti-DN-cadherin (1:1); anti-BrdU (1:100); anti-Prospero (1:3) (Hybridoma

Bank); anti-Lola (1:75) (gift from E. Giniger); anti-Col (1:50) (Dubois, Enriquez et al. 2007);

anti-Talin (1:30) (gift from N. Brown (Brown, Gregory et al. 2002)); anti-αTubulin (1:100);

Sigma-Aldrich, T9026); anti-LacZ (Promega, 1:1000). Rabbit polyclonal antibodies: anti-

Wicked (1:500) (gift from J.R. Huynh); anti-Miranda (1:200) (Matsuzaki, Ohshiro et al.

1998); anti-Brat (1:200) (Betschinger, Mechtler et al. 2006); anti-H3P (1:200, Upstate

Bioscience); anti-LacZ (Capell, 1:5000). The BrdU (5-Bromo-2’-deoxyuridine) was

purchased from Sigma-Aldrich; phalloidin (Interchim) and TOPRO3 (Molecular Probes).

Secondary antibodies: goat anti-mouse Alexa-Fluor-488, Alexa-Fluor-546 and Alexa-Fluor-

647 (Molecular Probes, 1:500).

Fly strains

The following fly strains were used: Nts2 (Bloomington Drosophila Stock

Center)(Shellenbarger and Mohler 1975), Pcol85-Gal4;mCD8GFP(Krzemien, Dubois et al.

2007), Pdome-Gal4(Bourbon, Gonzy-Treboul et al. 2002), esg-Gal4(Micchelli and Perrimon

2006), esgGFP (gift from N.Bausek), vikingGFP(Morin, Daneman et al. 2001), UAS-PonGFP

(gift from F. Schweisguth (Lu, Ackerman et al. 1999)), 7054-Gal4 (GET DB), lz-Gal4;UAS-

GFP

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69

(gift from B. Augé), Neur-lacZ(Phillips and Whittle 1993), Gbe + Su(H)m8 (gift from Fuss

B.(Furriols and Bray 2001), Oregon R was used as wild type.

BrdU pulse-chase experiments.

BrdU incorporation and pulse chase experiments were made as in (Lee, Wilkinson et

al. 2006), modified as described : Briefly, Pcol85-gal4;UASmCD8GFP L2 larvae were fed

for 4 h with BrdU (1 mg/ml; Sigma-Aldrich) diluted in classical fly food; one half of larvae

was immediately processed for BrdU staining (pulse experiments) and the second half was

left growing without BrdU for 24h before staining (chase experiment). Larval lymph glands

were dissected, fixed and immunostained (mouse mono-clonal anti-BrdU antibodies) as

described in Krzemien et al., 2007, with the exception that LG were treated in 2N HCl for 30

min prior to primary antibody immunolabelling.

Mitotic index measurements.

For counting mitotic cells we used immunostaining for H3P. Following a two-hours egg-

laying period, Pdome-gal4;UASmCD8GFP embryos were let develop up to 54h, 72h, 96h

and 120h after egg laying (ael), at 25°C and before dissection and staining of larval tissues for

mitotic cells, using H3P primary antibodies. The LG were mounted between a slide and a

cover slide and subjected to confocal microscopy analysis (Leica SP5 microscope). Z-series

of confocal images (distance between each scan ~1.5μm) were acquired. The mitotic index

was measured as follows: the number of mitotic figures in a given lymph gland was counted

and divided by the total number of cells in this lymph gland. The total cell number was

estimated as follows: the average surface of each LG was divided by the per-cell surface and

multiplied by the number of cell layers in the LG. The LG surface was measured using the

ImageJ software and the number of cell layers was calculated from “z” optical sections, using

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the Imaris software. To be able to count the number of mitotic figures in the MZ versus the

CZ, we used LG from Pdome-gal4;UASmCD8GFP larvae. For measuring the mitotic index

following wasp parasitisation, either Pdome-gal4;UASmCD8GFP or Oregon L2 larvae were

placed during 2h in presence of the wasp Leptopilina boulardi and then let to develop a

further 6h before dissection, fixation and staining of the LG. The results obtained for the 2 fly

strains were pooled up (nr of infected larvae = 20, number of control larvae= 13) since no

strain-specific difference could be observed. The statistical analyses were made with use of t-

Student test, at p < 0.05.

Notch ts experiment.

Nts2 (Bloomington Stock Center), a thermo-sensitive allele of the Notch receptor,

behaves as wild type at permissive temperature (22°C) but is inactive at restrictive

temperature (29°C) (Shellenbarger and Mohler 1975). In order to determine the temporal

window of N requirement during larval hematopoiesis, Nts2 larvae were shifted from

permissive to restrictive temperature at different developmental time points, for intervals of

24h or 48h, before returning back to 22°C until L3. In this study, we visualized crystal cells

by treating the larvae for 10 min at 60°C, which results in the specific blackening of crystal

cells (Rizki T. M. 1980). After the treatment the larvae were fixed and the lymph gland were

dissected out. Larvae left at 29°C following the shift to restrictive temperature were used as

controls.

Clonal analysis.

For cell-lineage clonal analyses, I used the Flp out inducible system in which the

flipase recombinase (FLP) gene is activated under the control of hsp70 regulatory sequences

(Golic and Lindquist 1989) and the following cross: yw,hsFlp; Sp/Cyo virgin females X yw;

actin FRT y+ FRT Gal4 UAS GFP males. Heat shock induced recombination between the two

FRTs (Flipase Recombination Targets) leads to excision of a cassette containing y+ (a “stop”

sequence), thereby activating Gal4 expression under the control of a constitutively active

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actin promoter. This leads in turn to GFP expression in the cell where the recombination took

place and all of its progeny. Eggs were collected for 2h and let to develop at 18°C. A 20 min

heat shock at 37°C (in water bath) was applied at different times during embryonic/larval

development (20 different time points between 17 and 168h ael, see Fig. 36). After the heat

shock, the larvae were shifted back at 18°C for 6h before being moved to 25°C for further

development. Development at 25°C was necessary to obtain sufficient numbers of crystal

cells, which do poorly differentiate at low temperature (our own observation). LG from late

L3 larvae were then dissected and stained with antibodies specific for plasmatocytes and

crystal cells, P1 and proPO antibodies, respectively. Stained lymph glands were analysed by

confocal microscopy (Leica SP5 microscope). Series of images from “z” sections were

analysed in 3D with use of the Imaris software. This 3D analysis was the most convenient

method to discriminate between mixed (plasmatocyte and crystal cell) and monotypic clones.

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75

Results

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Introduction.

Studies on the cellular immune response to parasitisation have shown the critical role

of the PSC in controlling this response and more generally Drosophila larval hematopoiesis.

As described in the introduction, in third instar larvae the primary lobes of the lymph

gland are organised in two distinct zones, in addition to the PSC: a medullary zone (MZ)

containing hematopoietic progenitors that can be identified by the expression of domeless

(dome) and a cortical zone (CZ) containing differentiating plasmatocytes and crystal cells that

have left the MZ (Jung, Evans et al. 2005). In col1 mutant larvae, the PSC is lost and no

differentiation of lamellocytes is observed upon wasp infestation. In addition to these two

phenotypes an increased number of differentiating crystal cells are found in col mutant LG,

raising the possibility that the PSC plays a general role in controlling hemocyte homeostasis

(Crozatier, Ubeda et al. 2004).

The first part of my PhD was to follow up these observations and study in more details

the PSC function. Some of the results that I obtained have been included in the article entitled

“Control of blood cell homeostasis in Drosophila larvae by the Posterior Signalling Centre”,

published in Nature in 2007 (Krzemien, Dubois et al. 2007). I will first briefly summarise

these results before complementing with additional data obtained since the 2007 publication

and describing my recent work on the search for the stem cells and cell lineage studies in the

lymph gland.

The PSC is required to maintain a pool of prohemocytes in 3rd instar larvae.

Whereas in mid-third instar wt larvae the MZ occupies a large volume of the primary

lobes, as evidenced by dome-gal4/UAS-mCD8GFP expression (Fig.19a), very few, if any

GFP-positive cells are detected in col mutant LG (Fig. 19b). Expression of tep4, another gene

specifically expressed in the MZ (Irving, Ubeda et al. 2005), is also lost in col mutant larvae

(Fig. 19c,d), confirming loss of the MZ. This disappearance correlates with an increased

differentiation of both crystal cells and plasmatocytes, as detected by proPO and P1

expression, respectively (Fig. 19e-f). No difference in size or morphology of the MZ is

observed, however, between wt and col mutant larvae in second (L2) and early third instar

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Fig. 19 The PSC is required to maintain a pool of hematopoietic progenitors. (a) The MZ, identified by the expression of a membrane–targeted GFP (dome-gal4/UAS-mCD8-GFP) in LG from third instar larvae, is lost in col mutant larvae (b). (c,d) The same result was obtained with tep4, another MZ marker. Conversely, the number of differentiating, pro-PO-positive crystal cells (a,b) and P1-positive plasmatocytes (e,f) is increased in col mutant LG. Topro markes nuclei;bar: 40μm

Fig. 20 Loss of col activity in the PSC does not affect the formation of the medullary zone in second instar larvae. (a) The MZ, identified by the expression of GFP (dome-gal4/UAS-mCD8-GFP) in LG from second instar wild-type larvae, forms normally in col mutant larvae (b).Topro marks nuclei. Bar: 40μm

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(EL3) (Fig20. a,b) indicating that the MZ forms independently of the PSC. We could

therefore conclude that the PSC activity is not necessary to establish the MZ but to maintain a

pool of hematopoietic progenitors in the MZ throughout larval development.

The same phenotype that is observed in col1 mutants is observed following selective

killing of PSC cells via targeted expression of the pro-apoptotic gene reaper (rpr), thus

strengthening the conclusion that communication between the PSC and MZ cells controls

larval hematopoiesis and that Col plays a major role in PSC activity (Fig. 21 a-c).

PSC specific expression of UAS transgenes was possible thanks to a transgene,

Pcol85-gal4 (made by gene replacement from a PlacZ insertion into the promoter of collier)

designed and obtained by L.Dubois. Pcol85gal4 driven expression of the reporter gene lacZ

completely overlaps the expression profile of Collier in the PSC, from embryonic stage 16 up

to the end of larval stages (Fig 22a-cf).

Ser expression in PSC cells is involved in maintenance of col expression.

Although Notch (N) activity within the LG was shown to be required for the

differentiation of crystal cells, the role of Ser expression in the PSC, which depends upon Col

activity remained unknown (Lebestky, Jung et al. 2003; Crozatier, Ubeda et al. 2004). To

determine which role Ser could play in the communication between the PSC and

prohemocytes, we targeted the expression of a dominant-negative form of Ser, SerTM (Sun

and Artavanis-Tsakonas 1996) to the PSC. This resulted in a decreased level of tep4 in the

MZ and increased differentiation of doxA3 positive cells (crystal cells), but not of P1 positive

cells (Fig. 23 c,d) indicating that the down-regulation of Ser signalling partly overlaps the col

mutant phenotype. Further analysis showed that PSC-targeted expression of SerTM results in

nearly complete loss of col transcription in the PSC of third instar larvae. A similar down-

regulation was also observed in Nts mutants, suggesting that maintaining high levels of col

transcription in the PSC requires N signalling mediated by Ser (Fig. 23a,b). This observation

is reminiscent of the regulatory loop involving Delta/N and proneural genes that operates

during lateral inhibition. Decreased col transcription is not due to a reduced number of PSC

cells, which can be followed by GFP labelling (data not shown).

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Fig. 21. Killing the PSC mimics the col1 phenotype Selective killing of PSC cells via targeted expression of the pro-apoptotic gene reaper (rpr) (Pcol85-Gal4/UAS-rpr) mimics the col1 phenotype; no PSC (a), more crystal cells (b) and no lamellocytes upon parasioid wasp infestation (c). Bar: 40μm

Fig. 22 Pcol85-Gal4 reproduces Col expression pattern in the lymph gland (a) A schematic representation of the structure of the collier gene and position of the Pcol85-gal4 insertion that is located ~600bp before the transcription start. (The rectangles symbolise the exons)(after Crozatier et al. 1999, modified).(b, c, c’, c’’) Confocal imaging of the PSC from Pcol85-gal4/UAS-lacZ third instar larvae stained with anti-lacZ and anti-Col anti-bodies. There is a cell to cell overlap between Col and Pcol85-gal4 driven expression of LacZ in the PSC. Bar: 20μm

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Ectopic expression of col in the MZ inhibits hemocyte differentiation

col is initially expressed in all embryonic LG precursor cells before becoming

restricted to the PSC at the end of embryogenesis (Crozatier, Ubeda et al. 2004). To address

the question whether this restriction is necessary for normal hematopoiesis, we ectopically

expressed Col in MZ cells (domeless-gal4/UAS-GFP/UAS-col). Staining for markers of

plasmatocytes and crystal cells revealed a virtually complete absence of hemocyte

differentiation in the third instar LG. Correlatively, most LG cells still stained for mCD8GFP

and tep4, indicating that Col expression in MZ cells prevents prohemocytes from

differentiating (Fig. 24 a,b).

In summary, experiments involving both loss-of-function and ectopic expression of

Col lead to the conclusion that the control of hemocyte homeostasis requires communication

between two types of cells : PSC cells that segregate in late embryos and maintain high levels

of Col expression throughout larval development and a pool of hematopoietic precursors that

do not express Col. They suggest that the main role of the PSC is to act, non cell

autonomously to prevent the activation of expression of hemocyte differentiation genes,

thereby maintaining a pool of multipotent precursors up to the end of larval devlopment.

The JAK/STAT pathway is activated in the MZ

JAK/STAT activity has been shown to promote dome expression via a positive

autoregulation in numerous tissues (Hombria, Brown et al. 2005; Arbouzova and Zeidler

2006). To assay whether dome-gal4 expression in the MZ reflected active JAK/STAT

signalling, we made use of an in vivo reporter of JAK/STAT activation, dome-MESO

(Hombria, Brown et al. 2005). Overlap between the cells expressing high levels of dome-GFP

and those expressing dome-MESO indicated that the JAK/STAT signalling pathway is

activated in MZ cells (Fig 25a,b). Similar to dome-GFP, expression of dome-MESO is

independent of col in L2 larvae but lost in L3 larval stage in col mutants, confirming that col

is required for maintaining the JAK/STAT pathway active in the MZ (data not shown).

To ask whether sustained JAK/STAT signalling was required to maintain

prohemocytes in the MZ, we analysed the LGs from Stat92E mutant larvae, STAT92E being

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a bdome>col dome>col

GFP+proPO+topro tep4

Fig. 24 Ectopic expression of col in the MZ inhibits hemocyte differentiation (a) Forced Col expression in the MZ (dome-gal4/UAS-GFP/UAS-Col) prevents progenitor cells from exiting the MZ and differentiating, (b) The MZ marker tep4 occupies almost the entire LG. Topro marks nuclei. Bar: 40 μm

col col

doxA3 doxA3

c d

a b

wt col>SerTM

**

Fig. 23 Ser in PSC cells permits maintenance of col expression. (a) col transcription, assayed with a probe which reveals the nascent transcripts, is decreased in the PSC (arrow) in Pcol85-gal4 /UAS-SerTM compared to wt (b) while transcription in adjacent pericardial cells (star) is unmodified.(c,d) the downregulation of col transcription correlates with an increased number of crystal cells. Bar: 40 μm

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the only nuclear effector of the JAK-STAT pathway in Drosophila. Nearly complete loss of

tep4 expression and increased hemocyte differentiation were observed in the primary lobes in

absence of STAT92E activity (Fig 26a-d). These results indicate that JAK/STAT signalling

activity is required for the maintenance of the MZ and the control of hemocyte production in

third instar larvae. The premature loss of MZ, together with increased differentiation of

hemocytes that are observed either in the absence of a functional PSC or upon loss of

JAK/STAT activity support the conclusion that PSC cells act in a non-cell-autonomous

manner to maintain JAK/STAT activity in the MZ cells, thereby maintaining their

prohemocyte character. We noted, however, that unlike in col mutant larvae, low levels of

tep4 expression could still be observed in stat mutant LGs, which correlates with

morphological evidence for a residual, only reduced MZ. Furthermore, loss of tep4 expression

and premature hemocyte differentiation in stat92E mutants were only observed in the primary

lobes, while secondary lobes maintained wt features. In contrast, in col mutants, premature

differentiation of crystal cells can be observed both in primary and in secondary lobes while

the primary lobes remaining spherical, a shape typical of LG of second instar larvae. All

together, these data suggested that the PSC performs functions in LG development in addition

to maintaining JAK/STAT signaling in the MZ. Yet, it appeared that JAK/STAT signalling in

the MZ is a key component of hemocyte homeostasis.

Wasp infestation switches off JAK/STAT signalling in the MZ and induces massive

differentiation of prohemocytes.

Egg-laying by parasitoïd wasps in Drosophila larvae triggers the production of

lamellocytes. Using antibodies raised against a lamellocyte-specific integrin α chain, α-PS4

(Crozatier, Ubeda et al. 2004; Irving, Ubeda et al. 2005), we confirmed the absence of

lamellocytes in unchallenged larvae. In contrast, massive differentiation of lamellocytes was

already observed 24h after wasp egg-laying. To our knowledge, lamellocytes are the only

Drosophila cells with a “cryptic” fate that is revealed only under specific conditions. (Fig.

27c,d). Strikingly, lamellocyte differentiation was paralleled by the premature loss of the

medullary zone (Fig. 27 a,b), indicating that the production of lamellocytes takes place at the

expense of the pool of prohemocytes which, during normal development persist until the

lymph gland bursts at metamorphosis. This reprogramming which bypasses the normal

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Fig. 26 JAK/STAT activity is required to maintain the pool of prohemocytes in the LG (a-d) In stat mutant LG, transcription of tep4 is almost completely lost from the primary lobe, correlating with an increased differentiation of doxA3-positive crystal cells compared to wt LG (c, d). Note that, in stat mutants, tep4 remains expressed in the posterior lobes (arrow), which do not differentiate crystal cells. Bar: 40μm

dome>GFP; dome-MEZO

GFP Anti-lacZ mergea a”a’

b b”b’

Fig. 25 JAK/STAT signalling pathway is activated in the MZ. (a,b) Expression of GFP (domeless-gal4/UAS-mCD8-GFP) and nuclear LacZ (dome-MESO) overlap in the MZ of third instar LG, indicating that the JAK/STAT signaling pathway is active in MZ cells. Residual Lac-Z staining in some cells that have left the MZ reflects the high stability of the β-galactosidase protein. Bar: 40μm

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hemocyte homeostasis revealed an unexpected level of plasticity of Drosophila

prohemocytes.

These new data led us to revise the model initially proposed in 2004 for the PSC

function (Fig. 12). Our present model, illustrated in Fig. 28 postulates that during normal

development, PSC cells act, in a non-cell autonomous manner, to maintain JAK/STAT

signalling activity in MZ cells in L3 larvae. The PSC activity, which depends upon Col, is

required in physiological conditions for preventing the premature differentiation of

multipotent prohemocytes into plasmatocytes and crystal cells. Notch signalling mediated by

Ser expression is required to maintain normal levels of col expression in the PSC. Following

wasp parasitisation, a signal likely emitted by plasmatocytes upon their detection of wasp

eggs is perceived by prohemocytes either directly or via the PSC, or both, which leads to

massive differentiation of prohemocytes into lamellocytes. In either case, Col activity in the

PSC is required for maintaining the competence of prohemocytes to adopt the lamellocyte

fate.

In this model we thus propose that the PSC acts as a microenvironmental niche for

prohemocytes.

PSC cells extend numerous filopodia.

In third instar larvae, the PSC is composed of about 30 – 40 cells per lobe. In addition

to a tight clustering, PSC cells show a particular morphology with the presence of numerous

cytoplasmic extensions that can be stained with an actin-GFP fusion protein (data not shown),

indicating that they are actin – based filopodia (Fig. 29). These filopodia are first observed in

early third instar larvae and progressively grow in size and branching complexity throughout

the third instar to extend at least over 2 to 3 cell diameters (Fig29 a-f). This raises the

intriguing possibility that direct cellular contacts between the PSC and a subset of MZ cells

could be mediated via filopodia and be important for the control functions exerted by the

PSC. Additionally, when observed in optical ‘z’ sections, the PSC appears to surround a

subpopulation of posterior MZ cells (Fig. 29 f-i). This tissue organisation raises the possibility

that the PSC establishes physical direct contacts with a subpopulation of MZ cells during the

third instar larval stage.

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Fig. 27 Premature differentiation of prohemocytes into lamellocytes upon wasp infestation. (a) The MZ identified by the expression of a membrane bound GFP (dome-gal4/UAS-mCD8-GFP) (b) is lost 24h after wasp infestation; (c,d) this parallels the massive induction of lamellocyte differentiation, detected by a-PS4 expression. Topro marks nuclei. Bar: 20μm

Fig. 28. A model of larval hematopoiesis in Drosophila. (a) During normal development, PSC cells (blue) act, in a non cell autonomous manner, to maintain JAK/STAT signalling activity in MZ cells (green area) in L3 larvae. This is required for preventing the premature differentiation of multipotent prohemocytes into plasmatocytes and crystal cells in the CZ. N signalling mediated by Ser expression in the PSC is required to maintain normal levels of col expression. (b) Reprogramming of prohemocytes in response to parasitisation : a signal likely emitted by plasmatocytes (grey circles) upon their detection of wasp eggs is perceived by prohemocytes either directly (S2) or via the PSC (S1->S2 relay), or both. In either case, Col activity in the PSC is required for prohemocytes to adopt the lamellocyte fate. Arrows indicate activation, vertical bars repression.

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Looking for stem cells.

The recent discovery that the Drosophila PSC functions as an hematopoietic niche in

maintaining a pool of uncommitted pro-hemocytes in the lymph gland up to the dispersal of

this hematopoietic organ at metamorphosis, raised the question of whether there exist

hematopoietic stem cells (HSC) in Drosophila, as there exists in vertebrates. Mouse HSCs

have first been operationally defined as able to long-term reconstitute hematopoietic tissue

after transplantation and give rise to the entire repertoire of hematopoietic cell types.

Unfortunately, a similar reconstitution assay is not currently available in Drosophila. More

general properties assigned to vertebrate HSC in the bone marrow include dependence of a

niche, multipotency, slow cycling, self-renewal, asymmetric divisions as a means to both self-

renew and give rise to a cell with differentiating capacities. In order to address the question of

whether a subset of cells in the medullary zone could be stem cells, I turned to some of these

criteria, using available molecular tools. First, I proceeded to test a series of “Drosophila stem

cell markers”, described under this broad term in the literature, reasoning that stem cells in the

lymph gland could possibly share cellular and molecular features with other stem cells.

One widespread stem cell characteristic is its capacity to divide asymmetrically in

order to produce a copy of itself and a second cell which either engages into differentiation or

represents an intermediate progenitor able to amplify itself before differentiation. Asymmetric

divisions can result from either the unequal segregation of intrinsic factors during mitosis, a

so-called “divisional asymmetry” or the influence of extrinsic cues, a so-called

“environmental asymmetry, or a combination of both (Wu, Egger et al. 2008) There are

examples of the two types of asymmetric division in the stem cell literature. One of the best-

described examples of divisional asymmetry is the division of Drosophila neuroblasts (NB) in

the larval central nervous system (CNS). In this kind of division, the determinants must be

distributed to one pole of the cell, while at the same time the mitotic spindle must rotate to

adopt the correct orientation, such that the axes of cell polarity and mitosis are parallel.

During cytokinesis, the determinants will be segregated into one daughter cell. Additionally,

the orientation of the mitotic spindle is crucial since, even if the determinants are distributed

to one pole of the cell, the axes of polarity and mitosis are orthogonal, the daughter cells will

have the same fate (Fig.30) (Macara 2004).

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Fig. 29 PSC cells extend the filopodia (a) The second instar larval LG stained with phalloidin (red) and Pcol85-gal4 /UAS-GFP (green) to outline the cell contours and the PSC, respectively. c,d, Expression of mCD8-GFP targeted to the PSC reveals the formation of cytoplasmic extensions, starting in early third instar larvae, (EL3, arrow); (b) such extensions are not detected at the second instar (L2). In third instar larvae (d) much longer cytoplasmic extensions are detected (green arrow); (e,f) PSC cells surround some MZ cells (domeMEZO-lacZ, red) forming a pocket at the posterior tip of the LG; (g,h) PSC cells stay usually in the contact with prohemocytes (domeMEZO-lacZ, blue).Topro marks nuclei. Bar: (e,g) 40μm, (a,d) 20μm, (b,c) 4 μm.

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During embryonic and larval neurogenesis, NBs undergo repeated asymmetric divisions, each

of which self-renews the NB and produces a smaller neural progenitor cell, called a ganglion

mother cell (GMC) that will itself undergo a single “differentiating division” to produce two

cells that exit the cell cycle and differentiate into either neurons or glial cells (Skeath and Thor

2003; Pearson and Doe 2004). NB divisions are not only morphologically but also molecularly

asymmetric and a series of intrinsic factors that control this asymetry have been identified

among which three proteins, Miranda – Brat – Prospero that segregate to the GMC. Miranda is

an adaptor protein responsible for localising Brat and Prospero at the cell cortex of the future

GMC. Brat controls the size of the newborn cell, in part by down-regulating d-Myc expression

in the GMC (Betschinger, Mechtler et al. 2006) and Prospero is a cell fate determining

transcription factor that represses NB-specific gene expression and activates genes for terminal

neuron differentiation (Betschinger, Mechtler et al. 2006; Choksi, Southall et al. 2006). Since

the expression patterns of Brat, Miranda and Prospero have been repeatedly used to characterise

asymmetric NB divisions, I tested whether any of these proteins was also expressed in the

lymph gland, using the larval CNS as a positive control tissue. I was not able to detect any

staining in the lymph gland (except two cells stained with Prospero antibody in one posterior

lobe) (Fig 31 a-f).

Among other markers of asymmetric divisions in the nervous system, I also used Pon-

GFP, a fusion to GFP of Pon (Partner of Numb), an adapter protein required for the proper

localization of Numb, an asymmetrically segregated regulator of Notch signalling during

mitosis (Lu, Rothenberg et al. 1998). Numb function was described both in the CNS (NB) and

peripheral nervous system (PNS) where the sensory organ precursor cells (SOPs) undergo

several rounds of asymmetric divisions to give rise to the various cells composing the external

sensory organ (Roegiers, Younger-Shepherd et al. 2001). I also used a reporter line, Neur-

LacZ reflecting the expression of Neuralised (Neur), an ubiquitin ligase contributing to

controlling the cell fate decisions imposed by Notch signalling (Le Borgne and Schweisguth

2003). As for other neural markers, the patterns of expression of Pon::GFP and Neu-lacZ did

not reveal any evidence for asymmetric division in the lymph gland, unlike observed in the

control CNS and PNS tissues (Fig. 31g-j).

Drosophila neural stem cells in the larval CNS can also be distinguished by their

large size and large nucleolus, as seen for example using the nucleolus marker Fibrillarin

(Betschinger, Mechtler et al. 2006). Anti-Fibrillarin staining of LG at different times during

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Fig. 30. Asymmetric and symmetric cell division (a) For asymmetric division, the cell determinants must be distributed to one pole of the cell and the mitotic spindle must then rotate into the correct orientation so that the axes of polarity and mitosis are parallel. During cytokinesis, the determinants will then be segregated into one daughter cell. This is the case for the NB asymmetric division. The protein complex Miranda-Brat-Prospero is adressed to the basal cortex of the dividing NB and segregates to the GMC. (b) If the determinants are distributed to one pole of the cell, but the axes of polarity and mitosis are orthogonal, the daughter cells will have the same fate (after Macara 2004, modified).

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Fig. 31. Stem cell markers in the lymph gland Numerous stem cells markers have been used, as indicated at each panel, to detect the potential presence of stem cells in the lymph gland. None of them gave evidence for stem cells being present in third instar larvae. The control tissues were CNS (b,d,f,h,p,r,v), eye disc (l,n,t) and wing disc (j) Topro3 is a nuclear marker, col>GFP marks the PSC and domeMEZO the MZ respectively. Bar 40μm, except CNS 80 μm.

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Table 1. Stem cell markers. This table summarises the stem cell markers used in my search for stem cells in the LG. The markers were chosen based on bibliographic sources and tested both in the lymph gland and control tissue, mostly the larval CNS. Different factors involved in asymmetric division and stem cell maintenance are listed.

93

the 3rd instar larval stage failed, however, to reveal any specific stem-cell like sub-population

of cells, based on this large nucleolus criteria (Fig. 31 u,v).

Finally, I tested some other candidate proteins such as Lola, CycE and Wicked (Fig.

31 q-t and not shown) (Betschinger, Mechtler et al. 2006; Davies E. 2007; Moch C. 2007), but

could not observe specific patterns of expression in the lymph gland. One should note

however, that most of these described stem cell markers were characterised for their role

and/or expression in tissues where a stem cell niche does not seem to exist. This is also the

case for example for Drosophila intestine stem cells (ISC), which nonetheless specifically

express Escargot, a transcription factor whose mRNA has also been used as marker for

Drosophila male GSCs (Kiger, White-Cooper et al. 2000; Micchelli and Perrimon 2006;

Ohlstein and Spradling 2006). I tested whether Escargot was specifically expressed in the LG

but either use of the reporter lines esg-gal4 and esg::GFP failed to indicate specific expression

of Esg in the LG (Fig. 31 o,p).

One well described example of environmental asymmetric division is the division of

the Drosophila male germline stem cells (GSC) to give rise to one GSC and one gonadial cell

that initiates differentiation (see introduction). Similarly, each Drosophila female GSC

divides asymmetrically to give rise to a GSC and a differentiated cystoblast. In either case, the

daughter cell which initiates differentiation is the cell, which gets displaced from the niche,

showing that the GSC character depends upon the maintenance of close contacts with the

niche. In Drosophila ovaries, GSCs maintain a direct contact with their niche via the

transmembrane protein DE-caherin, a situation reminiscent of the postulated (controversial)

role of N-cadherin in mediating direct contacts between vertebrate HSCs and osteoblasts (a

component of the vertebrate hematopoietic niche). Antibody staining of lymph glands for

either Drosophila DE-cadherin or DN-cadherin (the Drosophila N-cadherin homolog), did

not reveal, however, any specific pattern in the lymph gland tissue, (unlike described for E-

Cadherin by Banerjee’s group (Jung, Evans et al. 2005) (Fig. 31 k- n).

The environmental asymmetry of Drosophila GSC divisions is initiated by the specific

orientation of the mitotic spindle with respect to the niche-GSC axis. In a final attempt to see

whether cells localised close to the PSC did undergo oriented mitosis, I stained lymph glands

with both antibodies against the mitotic marker H3P (phosphorylated Histone3 Sauve,

Anderson et al. 1999) and the mitotic spindle marker α-tubulin (data not shown). My

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Fig. 32 Waves of mitosis in the lymph gland during larval devlopment. (a) The mitotic index, calculated as the number of mitotic figures/number of cells, is high in L2 and early L3 and drops drastically during the mid-to-late L3 period (c-f). (b) The majotity of mitotic figures (H3P staining, red) localise to the MZ (dome>GFP, green) throughout the entire LG development. ToPro3 marks nuclei. Bar: 40μm

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preliminary results did not reveal evidence for oriented mitotic spindles relative to the PSC,

but clearly more experiments need to be done to conclude definitely.

Another general property shared by many stem cells is a slow rate of cycling, a

property hypothesised to conserve their growth potential and reduce the risk of genetic injury

during mitosis. Cells with slow cycling time can be distinguished by DNA labelling

experiments, using nucleotide labels like bromodeoxyuridine (BrdU) which is incorporated

into the DNA during the S-phase. The BrdU incorporation is often used in a pulse-chase assay

in which the period of BrdU incorporation (pulse) by cells in S-phase is followed by a period

when BrdU is not supplied anymore (chase). After the chase period, cells are monitored for

their BrdU content. The slowly cycling cells retain BrdU while it is diluted in cells that divide

often. The “label retaining cells” method has led to the identification of stem cells in the

vertebrate kidney (Oliver, Maarouf et al. 2004), epidermis (Lavker and Sun 1983) or hair

follicle (Cotsarelis, Sun et al. 1990). I used the BrdU chase pulse experiment in order to

determine whether a population of slowly cycling cells did reside in the lymph gland,

focusing most of my attention on the cells in close proximity to the PSC. My preliminary

results suggest that there is no slow cycling cell population within the lymph gland.

In summary, I could not find evidence for the existence of stem cells in the lymph

gland, based on either criteria of asymmetric division, slow cycling or expression of specific

molecular markers of (Drosophila) stem cell character (Table 1). However, since negative

results cannot be considered as completely conclusive, I decided to address the potential

existence of stem cells in the lymph gland and the origin of the different types of hemocytes

using cell lineage analyses. As a first step on this line, I compared the patterns of mitoses and

differentiation in the developing lymph gland.

Waves of mitosis during development and after wasp infestation

In all developing organs, it is crucial for cells to decide between keeping proliferating

and undergoing differentiation. This decision is influenced both by cell intrinsic components

and extrinsic (environmental) cues (Lee and Orr-Weaver 2003). The lymph gland grows

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Fig. 33. The majority of hemocytes are post-mitotic. (a,a’,b) Differentiating crystal cells in the lymph gland are post-mitotic as shown by the absence of cells double positive for H3P and proPO. Many post-mitotic cells are observed in the CZ (c,d) and some few examples of dividing (P1 positive) plasmatocytes can be observed. (e) cells expressing lamellocyte marker are post-mitotic. Bar: 40 μm

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about about 160x between the end of embryogenesis and metamorphosis (from ~30 cells per

lobe in the embryo until ~5000 per anterior lobe in late 3rd instar larvae). This growth is due to

waves of mitosis occurring at the different larval stages during development (Sorrentino,

Carton et al. 2002). I analysed in detail the patterns of cell divisions in the lymph gland in

relation to differentiation, using co-immunostainings for H3P, a marker of mitotic cells, cells

of the medullary zone (dome>GFP) and markers of differentiating hemocytes while all nuclei

were stained with Topro.

I first found that the mitotic index (calculated as number of mitotic figures/number of

cells) is highest in L2 and early L3 larvae (Fig. 32 a, c-f), when the medullary zone grows and

contributes most of the lymph gland tissue (Sorrentino, Carton et al. 2002; Jung, Evans et al.

2005; Krzemien, Dubois et al. 2007). This index drops significantly between the mid and late

larval stages, when hemocyte differentiation takes place (Fig. 32 a). Correlatively, I found

very few mitotic figures corresponding to differentiating cells. Three-dimensional analysis of

lymph glands that were double-stained for H3P and either crystal cells (ProPO, or LzlacZ)

showed no double-positive cell, indicating that all the cells in the CZ which express crystal

cell differentiation markers are post-mitotic (Fig. 33 a,b). Although some dividing

plasmatocytes were only occasionally observed, they also seem to be largely post-mitotic

(Fig. 33c,d). These experiments allowed me to conclude that most cell divisions in the LG

take place in the MZ, i.e., when cells have kept a pro-hemocyte character, a conclusion

somewhat different from what was previously reported (Jung, Evans et al. 2005; Mandal,

Martinez-Agosto et al. 2007). Together, mitotic index measurements and the patterns of

mitoses in L2 and early L3 larvae suggested that the medullary zone may well represent a

transit amplification zone for immature prohemocytes. I was nevertheless intrigued to observe

that, starting early L3, a large number of mitotic figures localise to the periphery of the

medullary zone, as defined by dome>GFP expression, and thereby correspond to cells which

have left the MZ but do not yet express differentiation markers. This observation suggests that

there could exist a “subcortical zone” (SCZ) in the L3 lymph gland, reminiscent of the

subventrical zone (SVZ) in the vertebrate CNS, where at least a subset of progenitors could

undergo a terminal “differentiating” mitosis necessary to activate the differentiation program

(Martinez-Cerdeno, Noctor et al. 2006)

Premature, massive differentiation of pro-hemocytes into lamellocytes occurs as a

specific cellular immune response to wasp parasitisation. Although parasitic wasps such as

Leptopilina boulardi lay their eggs in second instar Drosophila larvae, likely when the larval

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Fig. 34. Parasitoid wasp infestation induce the increase of mitotic index in the LG. (a-c) The mitotic index of the LG rises ~2x 6h after parasitoid wasp infestation in comparison with control LG. Note the presence of low level of Col expression outside the PSC. This expression is lost 6h after wasp infestation (b,c).Bar: 40μm

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cuticle is soft enough, lamellocyte differentiation only takes place in third instar larvae,

indicating the importance of the L2-L3 transition to the development of the Drosophila

cellular immune competence (Sorrentino, Carton et al. 2002). It was previously reported that

lymph glands of parasitized larvae exhibit a clear increased in mitotic index in early L3. In

our experimental design (see materials and methods), this increase could be observed already

6 hours after infection, (in very early 3rd instar larvae) indicating that this burst of mitosis is

an immediate response to parasitisation and precedes by several hours the first signs of

lamellocyte differentiation which we could only detect 24 hours after wasp egg-laying

(Krzemien, Dubois et al. 2007), Makki et al. submitted) (Fig. 34)). To detect lamellocytes, we

previously used antibodies directed against the lamellocyte-specific α-integrin, αPS4

(Krzemien, Dubois et al. 2007). Since αPS4 antibodies were raised in rabbits, I had to turn to

another marker to be able to do double stainings with H3P. I chose Talin, a linker protein

connecting integrins with the cytoskeleton and which, similar to αPS4, is selectively

expressed in the lamellocytes (Brown, Gregory et al. 2002) and data not shown). Whereas no

Talin positive cell is present in absence of parasitisation, a large number of Talin-positive

lamellocytes is observed in lymph glands of parasitized larvae. Double staining for H3P and

Talin showed no overlap. The few mitotic figures than can be observed 24h post parasitisation

likely occur in the residual medullary zone (Fig. 33e). From this, I could conclude that the

Talin-expressing, differentiating lamellocytes in the lymph gland are post-mitotic cells. To

summarise, my results confirm that lamellocyte differentiation in response to wasp

parasitisation occurs at the expense of the pool of dividing progenitors that is observed during

normal larval development (Krzemien, Dubois et al. 2007). As previously described, this

differentiation is preceded by a burst of mitosis (Sorrentino et al., 2002). Since lamellocytes

are post-mitotic cells, it suggests that this post-parasitisation mitotic amplification

corresponds to a terminal, symmetric differentiative mitosis, allowing for the rapid, massive

production of lamellocytes that is observed after 24h.

Cell lineage analyses

The compared patterns of mitoses and differentiation in the LG indicate that the

medullary zone is a progenitor amplification zone – left aside the question of whether a

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Fig. 35. Working hypothesis for the hemocyte lineages in the LG Each diagram shows a hypothetical (working) model for hemocyte fate determination in the developing LG; In a) multipotent progenitors that can give rise to all three types of hemocytes: plasmatocytes (blue), crystal cells (orange), lamellocytes (L) and persist until the third instar larval stage. The cell fate decision is taken during the last cell division (differentiating mitosis), could result from asymmetric division, be stochastic, or depend upon environmental cues .e.g. Notch (N) signalling; b) Cell fate restriction of progenitors occurs early. In this situation, environmental cues may also play an important role (N); both models presuppose, that at early stages of development (L2) a large fraction of progenitors can be reprogrammed to become lamellocytes (L) upon infestation. The question of which type of precursors/progenitors can give rise to lamellocytes is open.

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subsets of cells are “stem cells”-, while the cortical zone is made of two types of

differentiating, mostly post-mitotic cells. Previous work by Jung et al., (2005) has shown that

cortical zone cells are derived from the medullary zone prohemocytes (Jung, Evans et al.

2005). My observation of many mitotic figures in an intermediate, subcortical zone suggests

that it corresponds to terminal, differentiative mitoses. The compared patterns of mitoses and

differentiation in the LG raises the question of when pro-hemocytes become committed to

differentiate in either a plasmatocyte or a crystal cell. Among several possibilities, one would

be that the decision to become a plasmatocyte or a crystal cell is concomitant with the

terminal mitosis in the SCZ (Fig. 35a). This decision could be predetermined (divisional

asymmetry), stochastic, or depend upon environmental cues, such as for example activation of

Notch signalling by Ser-expressing cells, a signalling which is required for pro-hemocytes to

adopt a crystal cell fate (Duvic, Hoffmann et al. 2002). Another possibility would be that

terminal divisions are symmetric and that restriction of cell fate is progressively imposed on

pro-hemocytes during the proliferation phase (Fig. 35b) as it occurs in vertebrate CNS

(Martinez-Cerdeno, Noctor et al. 2006). To discriminate between these and other

possibilities, I performed lineage analyses, taking advantage of the Flp-out technique (hs flp;

actinFRTstopFRT gal4UASGFP) (Golic and Lindquist 1989) that allows me to induce GFP-

marked clones at a given time during development and follow the fate and progeny of the

marked cells using immuno-staining specific for each hemocyte type. If the first hypothesis is

true, early and lately induced clones during LG development should contain both crystal cells

and plasmatocytes. If on the other hand the second hypothesis is right all late-born clones

would be monotypic (Fig. 35).

Clones were induced by heat-shock treatment of larvae, otherwise maintained at 18°C,

at different time points between 17h and 168h after egg laying (AEL at 18°C) i.e., between

the end of embryogenesis and the mid-L3 larval stage (Fig. 36). The larvae were then left to

develop until late L3 before dissection and immunostainings for crystal cells and

plasmatocytes using antibodies against proPO and P1, respectively (Fig. 37A). Mixed clones,

i.e., clones containing both crystal cells and plasmatocytes revealing multipotent progenitors

were only observed when clones were induced before 96h after egg laying, which corresponds

to the L1-L2 transition (Fig. 36, 37A a,b,e,f). Past this transition, only single cell type

(monotypic) clones were observed, indicating that from stage L2, the lymph gland contains a

mixture of progenitors already fated to give either plasmatocyte or crystal cell progeny and no

more bipotent progenitors (Fig.36,37A c,d). The frequency of lymph gland lobes containing at

least one mixed clone was significantly smaller in larvae submitted to heat-shock between 48-

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Fig. 36 Cell lineage analysis, using the Flp out technique (hs flp; actinFRTstopFRT gal4UASGFP). In order to obtain GFP+ clones, heat shock induced expression of the flipase, was applied, at various times during embryonic and larval development. The larvae were then let to develop until L3, when they were dissected and stained with plasmatocyte and crystal cell specific markers. The clones were analysed in term of hemocyte type content. The presence of the two types of hemocytes indicates the existence of multipotent progenitor while, monotypic clones correspond to the fate-restricted, unipotent precursors. The % of mixed clones correlates inversely with the developmental time, indicating that cell fate restriction occurs early.

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72h AEL (15%, n=32) than between 17 and 24h, 33%, n=41) indicating that fate restriction of

pro-hemocytes starts immediately post-embryogenesis (Fig. 36). Together, our observations

of the disappearance of common crystal cell/plasmatocyte progenitor after the L1-L2

transition and rise in mitotic index in L2 and early L3 supports the conclusion that the

medullary zone is a transient amplification zone of fate-restricted progenitors.

Further analysis of clones that were induced early showed large groups of clustered

plasmatocytes one one hand and heterogeneous clones containing only one or few proPO

positive crystal cells while the other GFP-positive cells of the clone expressed no hemocyte

marker. A mixed clone shown in Fig. 37A e,f, illustrates the difference in number that can

sometimes be observed between plasmatocytes and crystal cells in a single clone. These

observations suggest that plasmatocyte and crystal cell progenitors not only segregate early

but also display different mitotic capacity and program. Plasmatocyte progenitors seem to

undergo a high number of divisions before all progeny cells differentiate more or less

concertedly while the crystal cell progenitors seem to undergo fewer divisions before one or

few cells of the clones starts to differentiate. Whether these differences reflect the

involvement of Notch signalling in crystal cell and not plasmatocyte differentiation (Duvic,

Hoffmann et al. 2002; Lebestky, Jung et al. 2003) is addressed below (see discussion). These

differences could also reflect the fact that some differentiated plasmatocytes can still divide

while crystal cells are post-mitotic. careful observation showed numerous examples of two

crystal cells clusters, suggesting that they could originate from symmetric, differentiative

division of precursors selected from groups of crystal cell “competent” progenitors.

Lamellocytes and crystal cells share a common progenitor

While my analysis of hemocyte clones in the LG indicated that plasmatocytes and

crystal cells arise from separate progenitors that become restricted in cell fate before the L1-

L2 transition, the origin of lamellocytes which are induced to differentiate in L3 larvae

remained to be established (Fig. 36). Several possibilities could be envisaged, among which

the existence of a specific pool of lamellocyte progenitors/precursors expanding upon wasp

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Fig. 37A. Clonal analysis. (a, b) Early (17h ael) induced clone (GFP positive cells) containing both crystal cells (proPO) and plasmatocytes (P1); (c,d) small clones (2 cells) induced 8th days ael contain only one hemocyte type (arrows); (e,f) plasmatocytes differentiate in clonally related groups of cells, while crystal cells show a scattered distribiution; (g,h) a clone of PSC cells extending filopodia. PSC cells are clonally independent from hemocytes. Bar: 40μm.

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infestation or the re-programming of either crystal cell or plasmatocyte progenitors, or both.

To get further insight into this lamellocyte lineage question, I stained LG taken out from

larvae 24h and 30h after infestation for markers of two cell types simultaneously:

plasmatocytes and lamellocytes, using P1 and αPS4 antibodies, plasmatocytes and crystal

cells, using P1 and proPO antibodies or crystal cells and lamellocytes, using LzGFP and αPS4

antibody. Double staining for lamellocytes and plasmatocytes showed a complete absence of

overlap, indicating that these two types of hemocytes differentiate simultaneously in LG of

wasp-infested larvae, with no sign of common precursor (Fig. 38 a,b). Interestingly, P1

staining revealed that plasmatocytes display more numerous cytoplasmic extensions

following wasp infestation, than they normally do in healthy larvae (Fig. 38 c,d). This

suggests that, upon wasp infestation, plasmatocytes get “activated”, a term which I use by

reference to the activation observed for circulating plasmatocytes in response to injury,

without meaning that it is a similar process. Double staining for P1 and proPO also gave

completely non-overlapping patterns, as previously observed in healthy larvae (Fig. 38 c,d).

However, whereas in non parasitisized larvae the proPO positive cells display a crystal cell

morphology, including the presence of proPO crystals visible in light microscopy, I observed

that the proPO positive cells of parasitised larvae show a lamellocyte morphology (Fig.38 d*).

At the same time, I could not identify cells with crystal cell morphology, suggesting that

proPO expressing cells can differentiate as either crystal cells or lamellocytes, depending

upon the environmental conditions. To confirm that proPO positive cells switch from a crystal

cell to a lamellocyte fate in response to wasp parasitisation, I used another crystal cell specific

marker, lzGal4, enhancer-trap line wichh recapitulates lz expression in embryo and larvae

(Lebestky, Chang et al. 2000). The complete absence of GFP expression in

lzgal4;UASmCD8GFP 24h after infestation confirmed the (nearly) complete absence of

crystal cell differentiation in lymph glands otherwise full of differentiating, αPS4 positive

lamellocytes (Fig. 38e,f).

All together my data show that lamellocytes differentiate at the expense of crystal cells

from a common precursor. I therefore would like to propose that crystal cell precursors are re-

programmed to become lamellocytes upon wasp infestation. Unlike the scattered pattern of

crystal cells that is consistently observed in healthy larvae, including by clonal analyses,

lamellocytes are often found in clusters, suggesting a concerted differentiation program. Thus

the selection of crystal cell precursors among a group of competent progenitors that is

suggested by clonal analyses may not operate when these precursors adopt a lamellocyte fate.

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Fig. 37B Embryonic origin of secondary lobes. Clones induced during embryogenesis (within the first 24h of development at 18°C) are observed not only in primary lobes but also in secondary lobes (arrow), although, the development of secondary lobes is observed only since LL2, thus the secondary lobes seem to originate from the same pool of cells as primary lobes. Bar: 40μm

Fig. 38. No crystal cells differentiate in the lymph gland upon wasp infestation. Staining of the lymph gland for hemocyte-specific markers revealed the absence or drastic reduction in number of crystal cells following wasp infestation (c-f). Plasmatocytes are detected (a,b) but the majority of cells are lamellocytes that express the crystal cell marker proPO (c,d) The other crystal cell marker, Lz (lzgal4), is lost (e,f). Bar: 40μm

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Notch signalling and cell fate restriction

Serrate-mediated activation of Notch signalling has been shown to be instructive for

crystal cell formation (Duvic, Hoffmann et al. 2002; Lebestky, Jung et al. 2003). However,

these results did not give any idea about the temporal requirement of Notch signalling for the

crystal cell fate. Since my analysis of GFP labelled clones showed that the decision to become

either a plasmatocyte or a crystal cell is taken early during larval development while crystal

cell differentiation only takes place from mid-L3, I asked the question of whether N signalling

was involved in the early lineage decision or late differentiation program, or both. In order to

address this question, I repeated the Nts experiments (Nts is a temperature-sensitive mutant

allele of the N receptor), but restricting N inactivation to separate time windows during larval

development. As shown in Table 2, crystal cells form in conditions where N is inactivated for

either 24h or 48h during early larval development while they do not form when Nts larvae are

shifted to restrictive temperature in L3. These results confirm that N signalling is critically

required in L3 for crystal cell differentiation, in agreement with previous analyses of Ser

mutant clones (Lebestky, Jung et al. 2003). The question of whether it is also required for the

early plasmatocyte versus crystal cell restriction remains open because Nts experiments need

to be repeated in embryos maintained at 29°C up to early L3. Intriguingly, I observed an

increased number of crystal cells per LG when larvae were shifted to restrictive temperature

between 72h and 96h (corresponding to the L2 stage at 22°C), a period of mitotic

amplification of progenitors (data not shown). We infer from these results that N signalling

may already operate at that stage. In the absence of N activity, progenitors would keep

proliferating, resulting in an increased number of crystal cells if the larvae are shifted back

later to permissive temperature. Staining of LG from Nts larvae raised at restrictive

temperature and subject to wasp infestation confirmed a strongly reduced number of

lamellocytes, compared to control wt larvae, but they were not completely absent. It suggests

that N signalling may not be strictly required for bipotential lamellocyte/crystal cell pro-

hemocytes to adopt a lamellocyte fate per se, (this depends upon another, parasitisation-linked

signal (Krzemien, Dubois et al. 2007), Makki et al. submitted) and that the reduced

lamellocyte number rather reflects a more general role of N in the control of prohemocyte

proliferation.

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Table 2. Notch is necessary for terminal crystal cells differentation. The N signalling pathway could not be required for the plasmatocyte versus crystal cell fate decision, since we can still obtain crystal cells after N has been inactivated during the lineage restriction periode, however N is absolutely necessary for terminal crystal cell differentation. *Crozatier M. personal communication

Fig. 39. Expression pattern of collagen IV (Viking) in the lymph gland (A) The third instar larval LG is surrounded by the pockets of viking (VkgGFP). Groups of hemocytes seem to be enclosed in a kind of “pockets” of extra-cellular matrix (ECM). Within each of “pockets”, all the hemocytes seem to exhibit a similar behaviour, manifested in the level of JAK/STAT pathway activation (marked by domeMEZO-lacZ) or (B) expression of differentiation markers (plasmatocytes stained with anti-P1 antibody). (C) The PSC cells are also surrounded by ECM, as a group, not individually. Bar: 40μm

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The extracellular matrix of the lymph gland

Histochemical and cytological studies of Drosophila have demonstrated that basement

membrane and extracellular matrix surround internal organs that are in contact with the

hemocel. Collagen IV is a major structural component of basement membranes in the

developing fly and is synthesized mainly by hemocytes and fat body. Two collagen IV

molecules were indentified in Drosophila, Cg25C and Viking (Borchiellini, Coulon et al.

1996). The presence of Cg25C in the lymph gland was previously reported (Le Parco,

Knibiehler et al. 1986). Lastly the observation of Jung at et al. showed that Viking::GFP

(Vkg-GFP) is expressed in interesting pattern in the larval hematopoetic organ, where it forms

pockets surrounding groups of hemocytes (Jung, Evans et al. 2005).

I investigated in more details the Vkg-GFP expression pattern and I could observe that

indeed groups of hemocytes are surrounded by the collagen. Additionally it seems that all

cells within each group behave similarly. It is manifested e.g. in the synchronised inactivation

of the JAK/STAT pathway (monitored by dome mezo-lacZ reporter gene) or by the

expression of differentiation marker P1 (Fig.39 a,b). I could also observe that the PSC is

surrounded by the collagen IV which creates a well distinguishable pocket (Fig. 39a, c).

For the moment, the reason for the specific pattern of Viking accumulation is not

known. The exciting possibility would be that it is not only required as a physical support for

the lymph gland but also could be involved in the communication between the different cell

types.

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Discussion

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The PSC as a model of hematopoietic niche

Our model presented in 2007 for the control of larval hematopoiesis proposes that the

PSC plays the role of a microenvironmental niche for Drosophila prohemocytes, with

functional similarities to the hematopoietic stem cell niche in the vertebrate bone marrow

(Krzemien, Dubois et al. 2007). According to this model, PSC cells send signal(s) necessary

to maintain the JAK/STAT signalling in the medullary zone, which is itself required for

keeping prohemocytes in a non-differentiated state. Switching off JAK/STAT signalling is

indeed necessary for prohemocytes to differentiate. The nature of the signal(s) sent by PSC

cells remain(s) unknown, at both levels of its molecular character and the way it is supplied to

prohemocytes. We can envisage several modes of communication between the PSC and MZ

cells. First, it could be based on direct contacts between these two populations of cells. The

morphology of the lymph gland, where ~40 PSC cells occupy the posterior tip of the lymph

gland, suggests in this case that communication could only occur with a relatively small sub-

population of MZ cells. Another mode of communication, in which PSC could influence a

bigger population of MZ cells, would be to secrete a diffusible molecule able to reach long-

range targets. We can also imagine a third mode of signalling, that is the use of relay cells

which would be in direct contact with the PSC on one side and communicate with all the

other prohemocytes on the other. At the present time, none of these possibilities can be

formally excluded.

Because the PSC is required to maintain activity of the JAK/STAT in the MZ, the first

candidate for a signal provided by PSC cells was a ligand of the JAK/STAT pathway, one of

the cytokine-like Upds. In Drosophila, there exist three Upds: 1, 2 and 3. The results from

Rami Makki, another PhD student in our laboratory, indicate that Upd acting in the lymph

gland is Upd3. Upd3 expression was detected in the PSC and MZ (Makki et al. submitted), as

previously reported by Jung at al. for a upd3-gal4 line (Jung, Evans et al. 2005). Upd3

expression suggests that there could be both autocrine and paracrine delivery of Upd3 to MZ

cells. However, RNAi gene silencing experiments favour the autocrine mode, since

expression of upd3 RNAi in the PSC has no hematopoietic phenotype, while its expression in

MZ results in switching off the JAK/STAT signalling. upd2 mutant larvae do not show any

hematopoietic phenotype and Upd1 seems not to be expressed in the lymph gland (Makki et

al. submitted). These data seem to exclude Upds as a major signals sent by PSC, although

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experiments removing all three Upds from the PSC, which would exclude a possible

redundancy are now required to conclude definitively.

The observation that PSC cells extend filopodia raised the intriguing possibility that

they could be involved in direct cell-cell communication with the cells, which stay close to

the PSC. Staining with GFP-targeted to the membrane (Pcol85gal4; UASmCD8GFP) reveals

that PSC filopodia can reach 2-3 cell diameters. However, it remains possible that they are

longer, if the threshold of sensitivity reached in our staining and imaging conditions is a

limiting factor. The filopodia were previously shown to act in long-range signalling during the

formation of sensory organs in Drosophila. In this situation, the sensory organ precursors

(SOPs) which express Delta (N ligand) form filopodial extensions that mediate cell to cell

Notch signalling at several cells distance (De Joussineau, Soule et al. 2003). The fact that the

N ligand Serrate is expressed and functions (see below) in the PSC therefore left open the

possibility that filopodia could mediate “long distance” N signalling in the lymph gland.

Unfortunately, we were not able to observe activation of N signalling in proximity of the

PSC, using as a sensitive N read out, Gbe + Su(H)m8 reporter gene (Furriols and Bray 2001),

preventing any conclusion as to how the PSC communicates with MZ through N signalling

(data not shown). My attempts for interfering with the the formation of PSC filopodia did not

give conclusive results (data not show) and the function of these extensions remains open. In

any case, it is unlikely that filopodia, even if longer that we were able to detect, would be able

to reach all MZ cells when the lymph gland reaches its mature size in third instar larvae. One

alternative interesting possibility is that filopodia are required to increase the secreting surface

of PSC cells.

In an article published in the same issue of Nature as ours, Mandal et al. suggest that the

Hedgehog (Hh) signalling pathway is involved in the communication between the PSC and

MZ. These authors show that Hh is expressed by PSC cells, while downstream components of

the Hh signalling pathway, the receptor Patched (Ptc) and transcription factor Cubitus

interruptus (Ci) are expressed in the medullary zone; they also show that hhts mutant displays

a phenotype similar to that of col1 , which is a massive differentiation of hemocytes (Mandal,

Martinez-Agosto et al. 2007). A similar phenotype is also observed when a dominant-negative

form of Ci is expressed in the MZ, or in the absence of Antennapedia function which is

required for PSC formation. Mandal et al., thus propose that Hh expressed by PSC cells could

act in the lymph gland at long distance, as it was described in other Drosophila tissues

(Chuang and Kornberg 2000; Mandal, Martinez-Agosto et al. 2007) and be involved in

maintaining prohemocytes in non-differentiated state. Hh acts as a secreted morphogen.

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Diffusion of Hh from its localised source of expression forms a gradient of concentration,

resulting in the activation of different target genes, depending upon the distance from the

source (Kornberg and Guha 2007). In Mandal et al’s, article, no proposition of a target gene

has been made and although it is possible that Hh could act as a morphogen in the lymph

gland, it remains unknown how it could regulate the prohemocyte character. Further

characterisation of the role of Hh signalling in the LG is necessary.

While establishing the key role of the PSC in hemocyte homeostasis, our lab has

shown that a key read-out of PSC signalling is the maintenance of JAK/STAT activity in MZ

cells and Mandal et al. have proposed that one secreted signal originating from the PSC is Hh.

So far, however, the possible links between JAK/STAT and Hh signalling remain unknown.

The simplest hypothesis would be that Hh contributes to control the expression of

components of the JAK/STAT pathway in the MZ, such as for example Upd3 and/or

Domeless. It could explain why the phenotypes of col, antp, stat and hh mutants in the

lymph gland are so similar.

Neither the work from Mandal et al. nor ours brought evidence for the presence of

relay cells which would transform short-range signalling from the PSC into long-range

signalling in the entire medullary zone. For the moment, we do not dispose of markers

indicating the existence of specific subpopulations of cells in the MZ. Nevertheless, it remains

possible that some cells in direct contact with PSC and their filopodia could act as relay cells.

By looking at the distribution pattern of extracellular matrix components and particularly the

Collagen IV (Viking) in the lymph gland, I could observe that the PSC cells are surrounded

by a “pocket” of Viking::GFP (Fig. 39 A,C). It is very interesting in terms of signalling, since

ECM was shown to modulate spreading of different signals. Very recent data show that

Collagen IV directly participates in signalling between the niche and female GSCs. Collagen

IV, by binding Dpp, reduces the range of signal spreading from the source to only few GSCs

(Wang, Harris et al. 2008). Whether ECM could influence the spreading of PSC signals, is in

itself a question worth of persuing.

As one can see, too many gaps remain to draw a detailed model of the signalling

operating between the PSC and prohemocytes. This challenging question is now being

addressed in our laboratory by Delphine Pennetier, a PhD student, using different

experimental approaches, including transcriptome analysis of the lymph gland.

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Collier expression in the lymph gland, a matter of golden mean.

The PSC forms during late embryonic development. As described in the introduction,

Collier expression is first detected in all cells of the lymph gland anlage and its progressive

restriction to PSC cells occurs at around stage 14. This is a very important step since, as a

result, two groups of cells are defined, with one of them (PSC) controlling the behaviour of

the other one (MZ). As we have shown, the restriction of Col expression to PSC cells is

critical for proper hematopoiesis since, when conditionally expressed in the whole lymph

gland, Col almost completely blocks the differentiation of hemocytes. In summary, Col

activity while essential needs to be restricted to small cell population in the lymph gland.

We have now shown that maintenance of col expression in the PSC requires Serrate,

which our lab previously showed was a target of Col in the PSC, but whose function there

remained unknown (Crozatier, Ubeda et al. 2004). The phenotype that is observed when

expressing a dominant-negative form of Ser in the PSC is a decrease of col transcription and

an increase of hemocyte differentiation. We could infer from these observations that increased

differentiation is a secondary consequence of the loss of Col activity in the PSC, resulting in a

moderate col mutant like phenotype. However we cannot exclude that Serrate could have

other roles than maintaining col transcription, for example signalling to and instructing cells

in direct contact with the PSC cells. This question is quite difficult to address since Ser is both

downstream of Col and required to maintain Col expression, such that, the removal of one

influences the expression of the other. (Crozatier, Ubeda et al. 2004; Krzemien, Dubois et al.

2007).

Role of the PSC in immune response against the parasitoid wasps.

We know that Collier is necessary for the PSC function and that its activity is critical

in controlling the cellular immune response against the parasitic wasps since 2004, when a

lymph gland phenotype of col was first described (Crozatier, Ubeda et al. 2004). The model

proposed by Crozatier et al. at that time postulated that the PSC plays a role of a processing

centre, in integrating the information from the hemolymph and coordinating the behaviour of

prohemocytes. Briefly, this model proposed the following: in case of a danger (e.g. wasp

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parasitisation) the PSC senses an “alert message”, probably issued from circulating

plasmatocytes (S1) and, in response sends a new signal (S2) instructing the prohemocytes to

differentiate into lamellocytes (Fig. 12) (Crozatier, Ubeda et al. 2004). Today, we have to

revise this model, taking into account our new data. First of all, and as already largely

discussed above, the PSC is required in healthy larvae to maintain the JAK/STAT signalling

pathway active, through a non-cell-autonomous mechanism. We have therefore to postulate

that the PSC sends “a signal of maintenance” necessary to conserve a pool of non-

differentiated progenitors. In col mutant larvae, because this signal is lacking, all

prohemocytes undergo premature differentiation and this is probably why, upon wasp

parasitisation, there are no precursor cells left to differentiate into lamellocytes. Unlike the

previous model, our new model does not necessarily imply that the signal of infection, S1

(Fig. 28b), is perceived by the PSC. Signal S1 could as well over-ride the PSC maintenance

signal and provoke premature differentiation. In either case, the central conclusion brought by

our recent results is that there are two types of signals, one signal of maintenance for

competence issued from the PSC and one signal of danger released upon parasitisation. The

role of the PSC in maintaining non-differentiated prohemocytes is crucial for the cellular

immune response: the “cryptic lamellocyte fate” can only be triggered in undifferentiated pro-

hemocytes. The nature of the signal(s) over-riding the PSC function to provoke premature

differentiation of hemocytes and instructing differentiating hemocytes to become lamellocytes

is (are) a very intriguing, but for the moment non-solved question.

The PSC is not required for the initiation of JAK/STAT activation, since in col mutant

second instar larvae, the medullary zone appears to form correctly and dome-MEZO lacZ, the

read out of the pathway, is detected. We suspect however that the MZ cells of col are not

identical with wt in terms of competence and plasticity, since they seem to be unable to be

reprogrammed and differentiate into lamellocytes in response to parasitisation. As discussed

above, recent data have raised many new questions about the PSC-MZ communication. What

happens in MZ cells in col larvae? This is one of the many questions, which we hope can be

resolved, at least partially by transcriptome analysis.

It is now clear that the interactions between the PSC and the prohemocytes are

multilayered; on one hand, each new observation adds complexity, on the other hand it

reinforces the conclusion that the PSC is a true microenvironmental niche for haematopoietic

precursors. Interestingly, during the time of my PhD, it has been reported that, one vertebrate

ortholog of Collier, EBF2, is expressed in immature osteoblasts, a major “signalling”

component of the bone marrow endosteal niche. It is, however fair to say that drawing further

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A parallels between col function in the PSC and ebf2 in osteoblasts first necessitates a more

complete description of col and ebf2 function and targets in these cells.

Are there hematopoietic stem cells in the lymph gland?

When observed in optical ‘z’ sections, the PSC appears to form a cup surrounding a

small subpopulation of posterior MZ cells (Fig. 29 f-h). This tissue organisation suggests that

the PSC maintains physical direct contacts with a specific subpopulation of MZ cells during

the third instar larval stage. Whether these few cells could display hematopoietic stem cell

properties is an intriguing question. More generally, the question of the existence of the

hematopoietic stem cells in the Drosophila lymph gland is a very important one, but with, at

this point, no answer in the literature. The term “niche” given to the PSC somewhat implies

that it is a stem cell microenvironment and I decided to challenge the question of whether

there are multipotent haematopoietic stem cells in the Drosophila lymph gland, or only

progenitors lacking the stem cell properties of self-renewal and long life.

The stem cells were described both in Drosophila and vertebrate models, but although

much attention has been put on them recently, there is still no general definition of stemness.

At the same time, the stem cells exist in various tissues and the “definition” has probably to

be established for each individual type. We don’t know for instance if the hypothetic

Drosophila hematopoietic stem cells would resemble more Drosophila germline stem cells or

neuroblasts or may be vertebrate HSCs or rather have unique properties. Using available

markers, I tried to see whether in the lymph gland there exist cells, with at least some of

previously reported stem cell characteristics. It seems that the only property that is common to

all described stem cells is the capacity to undergo a self-renewing, asymmetric cell division,

either intrinsic or extrinsic. I therefore decided to first see if I could detect asymmetric cell

divisions in the lymph gland. Using several molecular markers derived mainly from the

characterisation of Drosophila neuroblasts, I could not get evidence for the existence of stem

cells in the lymph gland using the criteria of asymmetric division.

One difficulty in our search for stem cells in the lymph gland is that, aside molecular

markers, some stem cell characteristics seem rather specific of the stem cell type. For example

one of morphological trait of Drosophila neuroblasts is a big size and big nucleolus, which

contrasts with a much smaller size of the daughter GMC (Betschinger, Mechtler et al. 2006).

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On the contrary, the characteristics of HSCs isolated in vertebrates is a small size (Ratajczak

2008). As another example, Drosophila GSCs are attached to the niche through adherent

junctions and DE-cadherin, and it was believed until not long ago that similar tight contacts

were mediated by DN-cadherin between the vertebrate HSCs and their niche in the bone

marrow. However, recent data suggest that, in fact it is not the case (Kiel, He et al. 2007).

Discrepancies are also revealed when looking for the criteria of slow cycling cells using BrdU

pulse chase experiments. Many vertebrate stem cells were reported to retain the DNA

labelling, in support of a slow cycling rate (kidney, epidermis, hair (Lavker and Sun 1983;

Cotsarelis, Sun et al. 1990; Oliver, Maarouf et al. 2004). Recently however, it was shown that

this labelling is neither a specific nor a sensitive marker of HSC, since only a small fraction of

HSC retains BrdU in pulse chase experiment (Kiel, He et al. 2007). Keeping these

contradictory data in mind, I nevertheless tested the lymph gland both for the presence of cells

expressing either E- or N- cadherin, cells with a large nucleolus size and cells retaining BrdU.

I haven’t detected any signs of these characteristics in the lymph gland.

The negative results are always more difficult to interpret. Especially in our case, since

we try to detect a population of cells which, first, we don’t know if it exists and second we

don’t know where it could reside. Following our idea that the PSC acts as a

microenvironmental niche, I gave the most attention to the cells of primary lobes and

particularly to those in the proximity of the PSC during my analysis of stem cell markers.

However, other areas could be considered as well. For example, not much is known about the

function of the secondary lobes. It has been reported that they form before or at the L1-L2

transition (Sellin, Albrecht et al. 2006), a crucial period for the fate restriction of larval

hematopoietic progenitors (see below). One intriguing possibility is that multipotent

progenitors could be segregated to these secondary lobes, before fate restriction process in the

primary lobes and stay as “stem cells”, while primary lobe precursors would get restricted and

differentiate.

In a majority of tissues, stem cells constitute a very minor cell population and are

believed to be slowly cycling and quiescent. These characteristics make the stem cells

difficult to detect. Futhermore, the markers, which I used were mainly described in

neuroblasts or GSCs and it is possible that they are rather specific for these cells, while

hematopoietic precursors would have unique properties. In summary, although my

experiments did not reveal the presence of stem cell in the primary lobes of the lymph gland,

one cannot only rely on the expression of markers and the other stem cell characteristics

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mentioned above, to conclude that they do not exist. I felt that it was necessary to attack the

problem from another angle, using retrospective cell lineage analyses.

Proliferation versus differentiation

To explore the ontogeny of third instar larval lymph gland, I first decided to

characterise the precursors in both terms of their mitotic and differentiation behaviour. My

results confirmed that during the development of lymph gland, major mitotic waves occur

during second and early third instar larval stages when it grows significantly in size and

showed that most mitoses occur in the medullary zone.

The differentiating hemocytes in the CZ indeed turned to be mainly post-mitotic, with

the exception of a few dividing plasmatocytes. The ability of plasmatocytes to divide has

already been shown for embryonic plasmatocytes in circulation in the larval hemolymph

(Lanot, Zachary et al. 2001). What was disconcerting was to find most mitotic figures in the

medullary zone, because it contradicts results published by Jung et al., and Mandal et al.

Brought together with mitotic index measurements, it indicates that the medullary zone

represents a progenitor amplification zone, where progenitors increase in number. One

particularly puzzling observation was the significant number of mitoses taking place at the

border of the medullary and corresponding to cells which do not express anymore pro-

hemocyte markers but have not yet started to express differentiation markers. I termed this

area the subcortical zone (SCZ). This term of subcortical zone does not refer to a

morphological frontier or an expression of specific markers but rather to a cell status

somewhere between cycling progenitors and differentiated hemocytes. The MZ/SCZ/CZ

layered organisation of the 3rd instar lymph gland is reminiscent of that of the developing

cortex in the mammalian brain where there have been described two proliferative areas, the

ventricular zone (VZ) and subventricular zone (SVZ). Schematically, at early stages of

cortical development, before the SVZ forms, the VZ contains a mixture of neural stem cells,

the radial glial cells (RGC), which undergo self-renewing asymmetric divisions and

intermediate progenitors issued from this asymmetric division. During later stages of

corticogenesis, intermediate progenitors (IP) migrate to form the SVZ where they divide

symmetrically to either amplify the number of progenitors, generating two progenitors or

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form two post-mitotic neurons (Noctor, Martinez-Cerdeno et al. 2004; Martinez-Cerdeno,

Noctor et al. 2006; Noctor, Martinez-Cerdeno et al. 2008). Whether mitoses occurring in the

SCZ of the lymph gland are symmetric, differentiative divisions, is an appealing hypothesis

which now needs to be tested.

The cellular response to wasp parasitisation requires a rapid, massive production of

lamellocytes. Interestingly, I could observe that a wave of mitosis occurs already 6h after

wasp parasitisation, before any sign of lamellocyte differentiation (~24h post infestation).

This wave of mitosis could be required either for amplifying the number of lamellocyte

precursors or lamellocyte differentiation to occur, in case lamellocytes are issued from

differentiative -“lamellocytic” - divisions of pro-hemocytes, or both. If the peak of mitosis

observed 6h following parasitisation does indeed corresponds to symmetric, lamellocytic

divisions, it should lead to a depletion of progenitors from the medullary zone. This is

precisely what is observed when using GFP (dome>mCD8GFP) as a pro-hemocyte marker.

24h post parasitisation, there remains very few, if any progenitors in the LG (Fig. 27 a,b).

While comparing Collier expression between parisitized and control larvae, I made an

unexpected observation: Col is expressed at a low level in a large region of the anterior lobes

of non-infected larvae, in addition to its very strong expression in the PSC. 6h after wasp

infestation, however, Col expression is only detected in the PSC, indicating that the complete

loss of Col expression could correlate with the premature loss of the MZ (Fig.34 b,c).

Together with previous data showing that the forced expression of Collier in the medullary

zone prevents hemocyte differentiation, this correlation raises the possibility that premature

hemocyte differentiation following parasitisation could require complete switching off of Col

expression. The temporal order of events: switching off JAK/STAT signalling and switching

off Col needs however to be established, before concluding, whether the loss of Col

expression upon infestation is causative or merely a consequence of premature hemocyte

differentiation. It order to see if a low level of Col expression in the MZ is necessary for

normal hematopoiesis, we could either induce col mutant clones or express col RNAi in the

MZ and look at whether it results in a hematopoietic phenotype.

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The origin of larval hemocytes

The best way to trace back the origin and history of differentiated cells is to mark

precursors and follow their progeny until differentiation. A retrospective clonal analysis is a

bit like a time machine, which permits to look at how the precursor behaved before becoming

a differentiated cell. GFP positive clones were induced in the lymph gland at different

developmental times and the types of hemocytes differentiating within each clone were

determined by appropriate immunostainings in fully developed lymph glands.

Although very exciting, clonal analysis turned out to be complex and delicate. The

method demands a lot of setting up in order to obtain the adequate number of clones per

lymph gland lobe (not too many) that makes you confident that what you look at is a single

clone. Even though, it is still possible that cell migration could lead to a confusing situation

where seemingly separate clones originate from a single recombination event. Keeping this in

mind I was careful while analysing data using Flp-out system and I think that at least

preliminary conclusions can be drawn from this analysis.

The results from my cell lineage analysis suggest that, in early stages of larval

development (L1), the lymph gland contains a mixture of bipotent plasmatocyte/crystal cell

precursors and monotypic progenitors. With time, the population of bipotent progenitors

disappears and fate restriction is imposed progressively, between L1 and L2. When

considering together mitotic index measurements and clonal analyses, it seems appropriate to

conclude that the medullary zone in L2 and early L3 larvae represents an amplification zone

for restricted progenitors and does not contain stem cells with multipotent lineage capacity.

Of course, it cannot be excluded that our stochastic clonal analysis could have missed stem

cells if their number is very low and if they are quiescent, but we have no indication in

support of stem cells.

In summary, cell fate restriction of progenitors takes place along with larval

development, before a period of intense proliferation, followed by differentiation. Whether

the mitotic figures in the subcortical zone correspond to ultimate differentiative divisions of

intermediate progenitors on the way to terminal differentiation in the cortical zone should be

definitely established by BrdU pulse-chase experiments.

The mechanism of fate restriction of prohemocytes is still a mystery. It is unknown

what signal limits the potency of precursors to a specific fate and where it comes from. One

suspected mechanism was Notch signalling because it was previously reported that N is

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necessary for crystal cell fate differentiation (Lebestky, Chang et al. 2000; Duvic, Hoffmann

et al. 2002) but no data indicated the period when this signalling is required. To look at the

temporal involvement of N in crystal cell formation, we used shifting of Nts larvae to the

restrictive temperature, followed by analysis of the number of crystal cells differentiating in

L3. We could confirm that N is necessary during the period of terminal differentiation;

however the question of its possible involvement into the plasmatocyte versus crystal cell fate

restriction in early larvae is still open. This question definitely needs further investigation

since contradictory results have appeared in the literature about the role of N in the

specification of crystal cells in the embryo (Lebestky, Jung et al. 2003; Bataille, Auge et al.

2005).

When discussing cell fate determination, we cannot only consider plasmatocytes and

crystal cell, since wasp parasitisation induces the differentiation of a third hemocyte type, the

lamellocytes. The lamellocye is a “cryptic fate” which is only revealed upon specific

parasitisation conditions. My data show that, after wasp egg laying, the only cell types

detected in the lymph gland are plasmatocytes and lamellocytes. Crystal cells are absent. This

stongly suggests that crystal cells and lamellocytes derive from a common precursor and that

lamellocytes differentiate at the expense of crystal cells after parasitisation. My observation of

cells expressing the crystal cell marker proPO, while displaying lamellocyte morphology,

reinforces this conclusion. The existence of a bipotential crystal cell/lamellocyte precursor

was already suugested by Col over-expression experiments in the lymph gland, where

lamellocytes differentiated in absence of wasp parasitisation, while in the same time a

strongly decreased number of crystal cells was noted (Crozatier, Ubeda et al. 2004).

Analysis of monotypic clones induced early during lymph gland development revealed

a distinct behaviour of plasmatocyte and crystal cell fated-progenitors. Plasmatocytes appear

to differentiate in clonally related groups of cells differentiating together, while crystal cells

show a rather scattered distribution in clones, (even if often found in pairs), with one or few

crystal cells among a group of non differentiated cells. The case of plasmatocytes suggests a

sort of community effect. By contrast, crystal cell differentiation seems to proceed after a

“selection” process. One possible hypothesis is that early fate restriction creates crystal cell

competence groups, from which crystal cell precursors are selected by N signalling, in a

process similar to lateral inhibition in the Drosophila CNS. The N selection could act at the

level of an intermediate progenitor still able to undergo symmetric division into two crystal

cells. Unlike crystal cells, lamellocytes differentiate in clusters (Fig. 38 b,f). Coming back to

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the idea of bipotential crystal cell/lamellocyte precursors, it suggests that all cells from a

crystal cell competence group could undergo concerted lamellocytic division, bypassing the

N selection process. This idea fits well with the observation that, even though in limited

numbers, lamellocytes differentiate in Nts conditions.

When I decided to invest myself into studies of hemocyte development, we had only

simple, intuitive models for the developmental history of prohemocytes and the three

hemocyte types in the lymph gland (Fig. 35). Today, based on results acquired over the past

3-4 years, we can propose a more detailed view of larval hematopoiesis, leading to new

hypotheses summarised in Fig. 40.

What more could we learn with clonal analysis?

Clonal analysis appeared to be a source of many interesting, sometimes unexpected

data. For example, I could observe clones which I believe, contain only PSC cells, based on

localisation, cell clustering and cell shape, including the presence of filopodia (Fig. 37A g,h).

This observation suggests that the PSC cells, which were proposed to segregate from the rest

of the LG cells during embryogenesis solely based on expression of specific markers and

reporter genes (Crozatier, Ubeda et al. 2004; Mandal, Martinez-Agosto et al. 2007), are

indeed clonally related and segregate from pro-hemocytes already in embryos.

Clonal analysis also revealed very interesting information about the origin of the

posterior lobes. In some cases when heat shock was applied in embryos, I found clones

covering part of both the primary lobe and a posterior lobe in L3 lymph glands (Fig. 37B).

This result indicates that the primary and secondary lobes derive from the same pool of

embryonic LG cells. It was particularly interesting to observe clones containing both PSC and

secondary lobe cells on the same side of the LG, suggesting that secondary lobes could be

also “seeded” by PSC cells. A deeper analysis of such mixed clones, for example for Col

expression is extremely important since there exist almost no data concerning the origin of

secondary lobes. As mentioned above, work of Sellin et al. suggests that secondary lobes are

generated by splitting off the posterior part of anterior lobes in L1 larvae or at the L1-L2

transition (Sellin, Albrecht et al. 2006). The idea that this splitting off could segregate at the

same time multipotent progenitors and niche cells in a secondary hematopoietic site is an idea

worth pursuing.

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Concluding remarks.

The results which I obtained during my PhD work represent only one step on the way

to describing and understanding the control of Drosophila larval hematopoiesis and origin of

larval hemocytes. Not surprisingly, the new data that I acquired, while improving our

knowledge of the complex process of hematopoiesis in insects, raised at the same time many

new intriguing questions, especially about the nature of interactions brought into play by the

different cell types in the lymph gland.

The ongoing discussions in the literature concerning the mode of interaction between the

hematopoietic stem cells and their niche in the vertebrate bone marrow, especially the roles of

osteoblasts and endothelial cells which form the endosteal and vascular niche, respectively are

very influential. It is fair to say, however, that despite intense work over the last ten years, our

knowledge of the localisation of HSC in the bone marrow and their communication with the

complex niche is still fragmentary. It is not yet clear whether osteoblasts establish direct

contacts with HSCs or whether there may be intermediate soluble factors and even relay cells

(Kiel and Morrison 2008). Although we have not found evidence for bona fide stem cells in

Drosophila lymph gland, the LG thus remains a valuable model to investigate the possible

interactions between the niche and progenitor cells under its control. We certainly need to

remain cautious when comparing the PSC and the hematopoietic niche, and I don’t refer only

to the obvious differences between the functions and repertoires of Drosophila compared to

vertebrate hematopoietic cells. I rather refer to the origin of the niche cells. In Drosophila, the

PSC and hematopoietic progenitors derive from a common pool of cells, while in vertebrates

the definitive niche is contributed by cells having a distinct origin from that of hematopoietic

stem cells. The evolutionary diversification of cell communications controlling the ontogeny

and functioning of the cellular immune system is a fascinating question of biology.

One other point that I consider very important and worth being brought up at this point

in the discussion, is that we don’t know anything about hematopoiesis in the adult fly.

Available data suggest that there does not exist a hematopoietic organ in the imago and that

the circulating plasmatocytes in the adult hemolymph are embryo and larvae-derived (Lanot,

Zachary et al. 2001). However, I could observe that a fraction of the medullary zone (which

could still contain hypothetic stem cells), the PSC cells and posterior lobes are maintained in

the lymph gland up to metamorphosis, when this tissue disperses. We have absolutely no idea

of what the future of these cells is. One intriguing possibility would be that they are

maintained throughout the entire Drosophila life cycle, as it is the case for some stem cells

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described in other Drosophila tissues or vertebrates and seed some secondary hematopoietic

sites engaged in adult hemocyte formation.

As one can see, new answers open new questions and probably the future will bring us

more exciting information about the mysteries of hemocyte and blood formation.

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Résumé, en Français. L’hématopoiése larvaire de la drosophile a lieu au sein d’un organe spécialisé, la glande de la lymphe (LG) qui produit des plasmatocytes spécialisés dans la phagocytose et des cellules à cristaux nécessaires à la mélanisation des corps étrangers (4). La LG est aussi à l’origine des lamellocytes nécessaires à l’encapsulation de gros corps étrangers et qui se différencient en réponse à des challenges immuns particuliers tels que le parasitisme par des hyménoptères. En 2004, notre laboratoire a montré que Collier (Col), l’orthologue du facteur de transcription mammifère Early B-Cell Factor est exprimé et requis dans un petit groupe de cellules spécialisées de la LG, le PSC, pour la différenciation des lamellocytes en réponse au parasitisme. Outre le PSC, la LG est organisée en deux zones distinctes : une zone médullaire (MZ) contenant les cellules précurseurs et une zone corticale (CZ) formée des cellules différenciées ayant quitté la MZ . Au cours de la première partie de ma thèse, j’ai étudié le contrôle de l’hématopoièse larvaire et montré que le PSC régulait de manière cellulaire non autonome, l’activité de la signalisation JAK/STAT dans les cellules précurseurs, empêchant ainsi leur différenciation prématurée et préservant leur capacité à se différencier en lamellocytes. Le rôle clef du PSC dans le maitien d’un pool de progéniteurs l’apparente à la niche hématopoiétique des vertébrés, un micro-environnement cellulaire requis pour le maintien de cellules souches tout au long de la vie adulte. Ceci posait la question de l’existence de cellules souches hématopoiètiques chez la drosophile, une question que j’ai abordée dans la deuxième partie de ma thèse. L’utilisation de marqueurs de cellules souches, de marqueurs de division asymétrique, ou la recherche de cellules quiescentes ne m’ont pas permis d‘identifier des cellule souches dans la LG. J’ai alors mis en oeuvre des expériences de lignage cellulaire et montré que les pro-hémocytes acquièrent un destin plasmatocyte ou cellule à cristaux dés le premier satde larvaire, avant une phase de prolifération intense. Mes expériences ont révélé l’existence d’une région «sous-corticale » (SCZ) riche en divisions cellulaires, suggérant que les cellules quittant la MZ se divisent avant de se différencier. En conclusion, les résultats acquis au cours de ma thèse montrent que le PSC agit comme une niche en maintenant la présence d’un pool de précurseurs hématopoiètiques pré-déterminés tout au long du développement larvaire de la drosophile. Abstrakt po polsku. Organ hemetopoetyczny u Drosophila melanogaster nazwany został gruczołem limfatycznym (GL). Na początku przeobrażenia larwy w poczwarkę, ulega on rozpadowi i uwalnia do hemolimfy dwa typy hemocytów: plazmatocyty, odpowiedzialne za fagocytozę oraz komórki krystaliczne, niezbędne w procesie melanizacji ciał obcych. GL produkuje rownież trzeci typ hemocytów, lamellocyty, odpowiedzialne za proces enkapsulacji ciał obcych, zbyt dużych by mogly byc sfagocytowane przez plazmatocyty. Komórki te różnicują się jedynie w odpowiedzi na specyficzene immunologiczne wyzwanie, jakim jest atak pasożytniczych os, składających jaja w ciele larwy. W 2004, nasze laboratorium pokazało, iż gen collier (col), ortolog Early-B-Cell Factor wystepującego u kregowców, ulega ekspresji w małej podgrupie komórek GL, zwanej Tylnim Centrum Sygnalizayjnym (z ang. Posterior Signalling Center - PSC). col jest niezbędny do prawidłowego funcjonowania PSC oraz do różnicowania się lamellocytów w odpowiedzi na pasożytnictwo. Poza PSC, w gruczole limfatycznym, można rozróżnic obszar medullarny (medullary zone - MZ), zawierający hemocyty niezróżnicowane, czyli prohemocyty, oraz obszar korowy (cortical zone - CZ), zawierający zróżnicowane hemocyty. W pierwszej części mojej pracy doktorskiej, zainteresowałam się homeostazą hemocytów w gruczole limfatycznym, i pokazałam, iż PSC kontroluje równowagę pomiędzy pulą prohemocytów oraz zróżnicowanymi hemocytami. PSC wysyła sygnały do prohemocytłw, dzięki którym utrzymuje aktywość scieżki sygnalizacyjnej JAK/STAT, zapobiegając ich różnicowaniu i utrzymując je w stadium prekursorowym, niezbędnym do zróżnicowania lamellocytów w przypadku pasożytnictwa. Kluczowa rola PSC, działającego jako mikrośrodowisko dla prohemocytów, przywodzi na myśl niszę komórek macierzystych w szpiku kostnym kręgowców. Zainspirowana tym skojarzeniem, w drugiej części doktoratu, zadałam sobie pytanie, czy hematopoetyczne komórki macierzyste wystepują w GL. Jednakże na podstawie użytych znaczników komórek macierzystych, znaczników asymetrycznych podziałów komórkowych oraz wolno dzilących sie komórek, nie byłam w stanie strwierdzić obecności komórek macierzystych w GL. Dzięki analizie klonalnej, udało mi sie ustalić, że progenitory hemocytów, zostają ukierunkowane do specyficznej linii (plazmatocytearnej lub komorek krystalicznych) po koniec pierwszego stadium larwalnym. Po okres restrykcji, następuje okres intensywnego namnażania ukierunkowanych prekursorów. Dystrybucja mitoz w GL, sugeruje iż pomiędzy MZ i CZ wystepuje obszar podkorowy (subcortical zone – SCZ), gdzie prekursory mogłyby podejmować ostateczny, różnicujący podział komórkowy. Zaobserwowałam również, iż komórki krystaliczne i lamellocyty, wywodzą się prawdopodobnie ze wspólnych prekursorów.