Asymmetric cell division in the drosophila embryonic neuroblast

27 FIGURE 5.A MODEL FOR MITOTIC SPINDLE ORIENTATION AND ASYMMETRIC CELL DIVISION DURING MAMMALIAN NEUROGENESIS... 84 4.8 Normal cell cycle progression in mitotic NBs requires genetic int

THE NOVEL PROTEIN SPINDLE MIRANDA

INTERACTS WITH INSCUTEABLE

TO REGULATE DROSOPHILA

NEUROBLAST ASYMMETRIC CELL DIVISION

LEE SIEW CHING JOAN

B APPL SCI (HONS.), NUS

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

ACKNOWLEDGEMENTS

I would like to thank my supervisor, A/P Yang Xiaohang; for guiding me

throughout the course of my project, present members of the YXH lab; Drs Wang

Huashan, Lin Shuping and Yin Yijun; for helping me to troubleshoot problems that I

faced, Ms Lee Chai Ling; for assisting my project by making fly food and ordering

materials and members of my Thesis Advisory Commitee; A/Ps Tang Bor Luen, Li

Baojie and Cai Yu; for providing me useful suggestions during my meet-ups with

them I would also like to extend my appreciation to past and present staff of the NUS

Graduate School for Integrative Sciences and Engineering, Prof Ren Ee Chee and Ms

Hazlina Umar; for their administrative support during my candidature I am grateful

to my parents and all my friends, for motivating me to persevere in my research

LIST OF ABBREVIATIONS

ACD Asymmetric Cell Division

AGS Activator of G protein signaling

A-P Anterior-Posterior

APC Adenomatous polyposis coli

APC/C Anaphase Promoting Complex/Cyclosome

aPKC atypical Protein Kinase C

ASIP Atypical PKC isotype-specific Interacting Protein

Aur-A Aurora A kinase

BCIP 5-bromo-4-chloro-3-indoyl phosphate

BMP Bone Morphogenetic Protein

C elegans Caenorhabditis elegans

CIP Calf Intestinal Phosphatase

CNS Central Nervous System

D melanogaster Drosophila melanogaster

DNA Deoxyribonucleic acid

E coli Escherichia coli

EDTA Ethylenediamine tetraacetic acid

EGFR Epidermal Growth Factor Receptor

F-actin Filamentous actin

Fz Frizzled

GDI Guanine nucleotide dissociation inhibitor

GFP Green fluorescent protein

GMC Ganglion Mother Cell

GST Glutathione S-transferase

Gαi α subunit of heterotrimeric G protein

Gβ13F β subunit of heterotrimeric G protein

Gγ1 γ subunit of heterotrimeric G protein

LB Luria Bertani

Lgl Lethal giant larvae

Mud Mushroom body defective

Myo II Non-muscle myosin II

PAGE Polyacrylamide gel electrophoresis

PAR Partitioning deficient

PBS Phosphate Buffered Saline

PBT PBS + 0.1% Triton X-100

PCM Pericentriolar Material

PCR Polymerase chain reaction

PDZ PSD-95 Disc large ZO-1

Pins Partner of Inscuteable

PMSF Phenylmethylsulphonyl Fluoride

PNS Peripheral Nervous System

Prox1 Prospero-related homeobox 1

RGC Radial glial cell

SDS Sodium Dodecyl Sulphate

SOP Sensory Organ Precursor

Spim Spindle Miranda or CG9646 protein

LIST OF FIGURES

FIGURE 1.ACD IN THE C ELEGANS ONE-CELL ZYGOTE 8

FIGURE 2.ACD IN THE D ROSOPHILA NB 20

FIGURE 3.ACD IN THE D ROSOPHILA SOP CELL 24

FIGURE 4.ACD IN THE D ROSOPHILA MGSC 27

FIGURE 5.A MODEL FOR MITOTIC SPINDLE ORIENTATION AND ASYMMETRIC CELL DIVISION DURING MAMMALIAN NEUROGENESIS 31

FIGURE 6.MISLOCALIZATION OF MIRA AND DEFECTIVE SPINDLE ORIENTATION IS OBSERVED FROM RNAI EXPERIMENTS 57

FIGURE 7.GENERATION OF D964667 AND D964680 BY IMPRECISE MOBILIZATION OF P-ELEMENT INSERTION LINE EY(2)06560 58

FIGURE 8.RNA EXPRESSION PATTERNS OF SPIM 59

FIGURE 9.MIRA AND NUMB ARE LOCALIZED NORMALLY IN MITOTIC NBS IN SPIM MUTANT EMBRYOS 60

FIGURE 10.MIRA AND NUMB ARE MISLOCALIZED IN NBS UNDERGOING METAPHASE IN SPIM INSC 22(NM) EMBRYOS 62

FIGURE 11.IN SPIM INSC 22(NM) METAPHASE NBS,MIRA IS LOCALIZED AT THE MITOTIC SPINDLE AND RECRUITS PROS TO THE SPINDLE 63

FIGURE 12. SPIM INSC 22(NM) EMBRYOS DO NOT EXHIBIT HIGHER LEVELS OF APOPTOTIC CELL DEATH AS COMPARED TO WILD-TYPE EMBRYOS 65\

FIGURE 13.LGL IS ABSENT IN METAPHASE NBS IN SPIM INSC 22 (NM) EMBRYOS 67

FIGURE 14.MYOSIN II IS ABSENT IN METAPHASE NBS IN SPIM INSC 22 (NM) EMBRYOS 68

FIGURE 15.F-ACTIN IS NOT UNIFORMLY DISTRIBUTED AROUND THE CORTEX OF METAPHASE NBS IN SPIM INSC 22(NM) EMBRYOS 70

FIGURE 16. APKC IS ABSENT IN METAPHASE NBS OF SPIM INSC 22(NM) EMBRYOS 71

FIGURE 17.PINS IS ABSENT IN METAPHASE NBS OF SPIM INSC 22(NM) EMBRYOS 73

FIGURE 18.SUMMARY OF THE GENETIC INTERACTION OF SPIM WITH INSC AND THE CONCOMITANT EFFECT ON ASYMMETRIC PROTEIN LOCALIZATION 88

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

LIST OF ABBREVIATIONS iii

LIST OF FIGURES v

SUMMARY x

CHAPTER 1 INTRODUCTION 1 ASYMMETRIC CELL DIVISION 2

1.1 ASYMMETRIC CELL DIVISION IN CAENORHABDITIS ELEGANS 3

1.1.1 Establishment and Maintenance of the Anterior-Posterior (A-P) axis 3

1.1.2 Asymmetric spindle positioning 4

1.1.3 Asymmetrically segregated proteins 7

1.2 ASYMMETRIC CELL DIVISION IN DROSOPHILA MELANOGASTER 9

1.2.1 Drosophila Neuroblasts 9

1.2.1.1 Establishment and maintenance of NB apicobasal polarity 10

1.2.1.2 Coordination of mitotic spindle orientation with cortical polarity 11

1.2.1.3 Spindle asymmetry and differential cell size 12

1.2.1.4 Centrosome asymmetry 13

1.2.1.5 Mechanisms of segregating cell fate determinants 13

1.2.1.6 Cell cycle regulators and asymmetric protein localization 16

1.2.1.7 Role of cell fate determinants 18

1.2.1.8 Asymmetric cell division and tumour suppression 19

1.2.2 Drosophila Sensory Organ Precursors (SOP) 20

1.2.2.1 Establishment of SOP planar polarity and Numb segregation 21

1.2.2.2 Directional signaling and cell fate difference 22

1.2.3 Drosophila Germline Stem Cells 25

1.3 ASYMMETRIC CELL DIVISION IN VERTEBRATES 28

1.3.1 Neural progenitor cells 28

1.3.1.1 Mitotic spindle orientation and modes of cell division 28

1.3.1.2 Segregating cell fate determinants 29

1.3.2 Epithelial cells 31

CHAPTER 2 MATERIALS AND MeTHODS 2.1 MOLECULAR WORK 34

2.1.1 Recombinant DNA methods 34

2.1.2 Strains and growth conditions 34

2.1.3 Preparation of competent E coli cells for heat-shock transformation 35

2.1.4 Cloning strategy and heat-shock transformation 35

2.1.5 PCR reaction 36

2.1.6 Plasmid DNA preparation 37

2.1.7 Protein analysis 38

2.1.8 Generation of polyclonal antibody 39

2.2 FLY WORK 40

2.2.1 Embryo fixing 40

2.2.2 Antibody staining 41

2.2.3 Antibodies 42

2.2.4 In situ Cell Death Detection 42

2.2.5 Confocal analysis and image processing 43

2.2.6 Whole embryo RNA in-situ hybridization 43

2.2.7 Double-stranded RNA interference 45

2.2.8 Single fly genomic DNA extraction 46

2.2.9 Mobilization of EP element 47

2.2.10 Generation of double mutants of insc 22 and CG9646 47

2.2.11 Removal of maternal contribution of CG9646 in embryos 48

2.3 Fly stocks used 49

2.4 Primers used 50

CHAPTER 3 RESULTS 3.1 BACKGROUND 53

3.2 RESULTS 55

3.2.1 Defects in Miranda mislocalization and spindle orientation are observed in RNAi experiments in insc mutant background 55

3.2.2 Generation of genetic mutants of CG9646 (Spim) 57

3.2.3 CG9646 RNA is expressed in neuroblasts 58

3.2.4 Mira and Numb are localized normally in spim mutant NBs 59

3.2.5 Mira and Numb are mislocalized in spim insc (nm) mutant NBs 60

3.2.6 Mira recruits Pros and is associated with the mitotic spindle 61

3.2.7 Embryos deficient in spim and insc are not undergoing elevated levels of apoptotic cell death 62

3.2.8 Lgl is absent in spim insc (nm) metaphase NBs 64

3.2.9 Non-muscle myosin II is absent in spim insc (nm) metaphase NBs 66

3.2.10 NBs deficient in spim and insc exhibit aberrant F-actin localization 69

3.2.11 Apical crescents of Pins are absent in NBs deficient in spim and insc 69

CHAPTER 4 DISCUSSION 4 DISCUSSION 75

4.1 Spim is regulated by the Sna family transcription factors and may be involved in telophase rescue of cell fate determinants in insc NBs 75

4.2 The Mira mislocalization phenotype in genetic mutants of spim insc differs from that obtained from spim knockdown in insc embryos 77

4.3 Spim is a novel protein and may interact with CG9986 and synapsin 78

4.4 Spim RNA is expressed in NBs 79

4.5 Maternal dosage of Spim masks the phenotype of spim insc mutants 80

4.6 Basal localization of Mira/Pros in NBs requires genetic interaction between

Spim and Insc 81

4.7 Pins expression in NBs by metaphase requires genetic interaction of Spim

and Insc 84

4.8 Normal cell cycle progression in mitotic NBs requires genetic interaction

between Spim and Insc 85

4.9 Normal embryonic development requires the genetic interaction between

Spim and Insc 87

CHAPTER 5 BIBLIOGRAPHY

5 BIBLIOGRAPHY 90

SUMMARY

Drosophila CG9646 protein, named as Spindle Miranda (Spim), is a novel

coiled-coil protein that was isolated from a microarray screen for potential effectors

of telophase rescue in insc mutant background Both Spim and Insc are positively

regulated by the Snail family of transcription factors Single mutants of spim do not

exhibit any defects in development or asymmetric division Only spim insc double

mutant embryos manifest abnormalities like delayed development and severe

mislocalization of cell fate determinants in NBs; Mira/Pros associates with the mitotic

spindle These findings strongly support the existence of a genetic interaction between Spim and Insc in the NB, as Insc is specifically expressed in NBs The

genetic interaction between Spim and Insc is involved in targeting Mira/Pros to the

basal cortex in mitotic NBs, possibly by stabilizing the actin cytoskeleton and interacting with Lgl and non-muscle Myosin II The Spim-Insc genetic interaction is

required for Pins expression and may also have role in cell cycle regulation and

development

Chapter 1 Introduction

1 ASYMMETRIC CELL DIVISION

Asymmetric cell division (ACD) is a highly conserved mechanism to generate

cellular diversity during development in multi-cellular organisms and is also an

attractive means for stem cells to balance the competing needs of self-renewal and

differentiation During ACD, the mother cell divides to generate two daughter cells

with different cell fates

A fundamental prerequisite for ACD is to establish an axis of cell polarity

Only in polarized cells can the mitotic spindle be correctly orientated and cell-fate

determinants localized asymmetrically These two events are critical and must be

highly coordinated to ensure the segregation of determinants into only one daughter

cell

Studies undertaken in the invertebrate animals Drosophila melanogaster and

Caenorhabditis elegans have led to the identification of complex molecular

machinery that polarizes cells and subsequently enables cell-fate determinants to be

differentially inherited by the two daughter cells, thereby allowing them to adopt

different fates (Gonczy, 2008; Knoblich, 2008) These studies have paved the way for

understanding the mechanisms and molecules of ACD in vertebrates, providing

increasing evidence that the fundamental principles and molecular players underlying

ACD appear to be conserved throughout metazoan evolution

1.1 ASYMMETRIC CELL DIVISION IN CAENORHABDITIS ELEGANS

The Caenorhabditis elegans (C elegans) one-cell zygote, the P0 cell, has been

particularly useful for dissecting the mechanisms of ACD (Figure 1) The zygote

divides asymmetrically to produce a larger AB cell fated to make ectoderm, and a

smaller P1 cell that produces mesoderm, endoderm and germ line in a series of

asymmetric divisions (Doe and Bowerman, 2001)

1.1.1 Establishment and Maintenance of the Anterior-Posterior (A-P) axis

Prior to sperm entry, a complex formed by the PDZ-domain proteins PAR-3

and PAR-6, as well as the atypical protein kinase C (PKC-3), is present throughout

the cell cortex (Etemad-Moghadam et al., 1995; Tabuse, 1998, Hung and Kemphues,

1999) Shortly after fertilization, the PAR-3–PAR-6–aPKC complex retracts towards

the anterior, along with the contractile actomyosin network (Cuenca et al., 2003) At

the same time, the ring-finger protein PAR-2 (Boyd et al., 1996) and the conserved

protein kinase PAR-1 (Guo and Kemphues, 1995) become enriched at the posterior,

where the actomyosin network is non-contractile Thereafter, the PAR-3–PAR-6–

PKC-3-containing domain retracts further, whereas the PAR-2-containing domain

expands reciprocally As a result, at the end of the first cell cycle, PAR-3–PAR-6–

PKC-3 covers the anterior and PAR-2 covers the posterior half of the embryo (Cuenca et al., 2003)

The anterior and posterior cortical domains are maintained as distinct entities

by various means In the absence of the 14-3-3 protein PAR-5, the two domains

become mixed (Morton, 2002) Moreover, reciprocal inhibitory interactions are important In the absence of PAR-3–PAR-6–PKC-3, cortical PAR-2 expands to fill

the entire circumference of the embryo (Boyd et al., 1996) Conversely, although

initially cleared from the posterior, PAR-3–PAR-6–aPKC is not restricted to the

anterior in par-2 mutant embryos (Cuenca et al., 2003) Reciprocal interactions also

occur between the actomyosin network and PAR proteins Thus, whereas

anterior-directed movement of the actomyosin network is essential for establishing distinct

cortical domains (Hill and Strome 1988; Guo and Kemphues, 1996; Shelton et al.,

1999), PAR proteins are needed reciprocally for proper actomyosin movements (Cheeks, 2004)

Components that are associated with the PAR proteins, such as CDC-37 and

CDC-42, help establish and maintain A–P polarity The chaperone protein CDC-37

primarily stabilizes PKC-3 (Beers and Kemphues, 2006) Severe depletion of the

small GTPase CDC-42 prevents PAR-6 cortical localization, whereas PAR-2 is present throughout the circumference of the embryo (Schonegg and Hyman, 2006)

As some cortical PAR-6 is present in embryos that have been simultaneously depleted

of CDC-42 and PAR-2, CDC-42 seems to promote cortical enrichment of PAR-6

(Gotta et al., 2001) partly by removing cortical PAR-2

1.1.2 Asymmetric spindle positioning

In the one-cell zygote, the centrally-located mitotic spindle is displaced asymmetrically as it elongates during anaphase, becoming positioned closer to the

posterior cortex by the end of mitosis

Experiments in which the spindle was severed in live specimens using a laser

microbeam revealed that pulling forces act on the two spindle poles, presumably

along astral microtubules that emanate from each centrosome and that abut the cell

cortex with their plus ends (Grill et al., 2001) Importantly, A–P polarity cues regulate

an imbalance of pulling forces, with a larger net force pulling on the posterior spindle

pole, thus explaining the asymmetric spindle positioning (Grill et al., 2003)

The pulling forces acting at the spindle are generated by the process of

microtubule depolymerization (Nguyen-Ngoc et al., 2007) A temperature-sensitive

allele of the β-tubulin gene tbb-2, which confers partial resistance to microtubule

depolymerization induced by cold or benomyl, exhibits impaired pulling forces

Moreover, preventing microtubule dynamics using taxol abolishes pulling forces

(Nguyen-Ngoc et al., 2007)

The minus-end-directed motor dynein is also important for pulling forces

Spindle-severing experiments following temperature shift of conditional mutant

alleles of the dynein heavy chain gene dhc-1, mild RNAi-mediated inactivation of the

associated lissencephaly protein-1 (LIS-1) or depletion of the

dynein-associated component roadblock (DYRB-1) all show marked reduction of pulling

forces (Nguyen-Ngoc et al, 2007; Couwenbergs, 2007)

In addition to microtubule depolymerization and dynein function, two partially redundant G-proteins, GOA-1 and GPA-16 (referred to collectively as Gα,

the essentially identical GoLoco-motif proteins GPR-1 and GPR-2 (referred to collectively as GPR-1/2) and the coiled-coil protein LIN-5 are required for asymmetric spindle positioning (Gotta and Ahringer, 2001; Colombo, 2003, Srinivasan et al., 2003) Although A–P polarity is not affected in embryos that have

been depleted of Gα, GPR-1/2 or LIN-5, pulling forces are essentially absent, resulting in symmetric spindle position and equal first cleavage (Colombo, 2003;

Nguyen-Ngoc et al., 2007) In C elegans, Gα, GPR-1/2 and LIN-5 are present in a

ternary complex that is enriched at the cortex (Lorson et al., 2000; Colombo, 2003;

Srinivasan et al., 2003) Available evidence suggests that LIN-5 interacts with GPR-1/2, which also associates with Gα

Gβγ is a negative regulator of Gα-mediated pulling forces, as forces increase

in embryos that have been depleted of Gβγ (Afshar, 2004) and are absent in embryos

that lack both Gβγ and Gα (Gotta and Ahringer, 2001; Tsou et al., 2003) Moreover,

Gα–GDP, not Gα–GTP, seems to be crucial for pulling forces, as the positive

regulator GPR-1/2 associates strictly with Gα–GDP (Colombo, 2003; Gotta et al.,

2003; Willard et al., 2004) The fact that overexpression of Gαi, but not of a mutant

that mimics Gαi–GTP, perturbs neuroblast asymmetric cell division (Schaefer et al.,

2001) is compatible with Gα–GDP being relevant for spindle positioning

The ternary complex of LIN-5–GPR-1/2–Gα affects cortical dynein DHC-1

co-immunoprecipitates the Gα proteins, whereas LIS-1 co-immunoprecipitates

GPR-1/2 and LIN-5 in a dhc-1-dependent manner (Nguyen-Ngoc et al., 2007)

Similarly, GFP-DYRB-1 co-immunoprecipitates GPR-1/2 and LIN-5 (Couwenbergs,

2007) Interestingly, LIS-1 interacts with LIN-5 even when GPR-1/2 is depleted,

whereas LIS-1 does not associate with GPR-1/2 when LIN-5 is depleted

(Nguyen-Ngoc et al., 2007) This suggests that the dynein complex interacts first with LIN-5

and then becomes associated with GPR-1/2, probably owing to the interaction between LIN-5 and GPR-1/2 Because GPR-1/2 in turn binds myristoylated Gα, this

could serve to anchor dynein at the cell cortex Accordingly, cortical dynein is

diminished when Gα, GPR-1/2 or LIN-5 are depleted (Nguyen-Ngoc et al, 2007)

Therefore, the ternary complex promotes recruitment of dynein to the cell cortex,

where it generates pulling forces on astral microtubules

Cortical anchoring of dynein by the ternary complex does not readily explain

why a larger net pulling force is exerted on the posterior side, as cortical dynein

distribution does not appear to be asymmetric (Gonczy et al., 1999; Nguyen-Ngoc et

al., 2007) Nevertheless, dynein activity could be modulated differentially on the two

sides There is more GPR-1/2 on the posterior cortex (Colombo, 2003, Gotta et al.,

2003), and so it has been speculated that GPR-1/2 might have a role in activating

dynein Appropriate assays for measuring cortical dynein activity must be developed

to investigate this and related possibilities

1.1.3 Asymmetrically segregated proteins

The generation of different daughter cell fates in the C elegans zygote relies

on the differential localization of a number of cytoplasmic proteins Two closely

related CCCH-finger proteins MEX-5 and MEX-6 are asymmetrically localized to the

anterior half of the zygotic cytoplasm (Schubert et al., 2000) MEX-5 and MEX-6

acts by restricting the localization of germline-specific proteins such as SKN-1,

PAL-1, MEX-PAL-1, POS-1 and the Zn-finger protein PIE-1 to the posterior end of the zygote

(Seydoux et al., 1996) In the absence of MEX-5 and MEX-6, germ-line specific

proteins, which are normally only inherited by the P1 cell, are expressed in both

daughter cells

Figure 1 ACD in the C elegans one-cell zygote The mother cell is polarized during

anaphase, and the distribution of different components important for polarity establishment, spindle positioning and cell fate determination is illustrated for the

mother cell, and the distribution of cell-fate determinants is shown for daughter cells

immediately after mitosis The figure is adapted from Gonczy (2008)

1.2 ASYMMETRIC CELL DIVISION IN DROSOPHILA MELANOGASTER

1.2.1 Drosophila Neuroblasts

Drosophila neural precursor cells, called neuroblasts (NBs), undergo a

stereotyped program of successive asymmetric divisions (Figure 2) Single cells within the presumptive neuroectoderm are selected to become NBs by a process

called “lateral inhibition” (Campos-Ortega, 1995) “Lateral inhibition” involves the

interaction of two phenotypicallyopposite classes of genes: the proneural genes, such

as achaete (ac), scute (sc) and lethal of scute (l’sc), and the neurogenic genes, such as

Notch and its ligandDelta and gene products of the Enhancer of split gene complex

E(spl)-C, which inhibit NB formation Once specified, NBs delaminate from the

neuroectoderm, and rotate their mitotic spindle 90° perpendicular to the epithelial

plane Subsequent NB divisions are thus oriented along the apico-basal axis (Kaltschmidt et al, 2000; Kaltschmidt and Brand, 2002)

Each asymmetric division in the NB gives rise to a larger apical neuroblast

that continues to divide asymmetrically and a smaller basal ganglion mother cell

(GMC), which divides terminally to generate two postmitotic neurons or glia Embryonic NBs divide up to about 18 times, decreasing in size at each division until

the end of embryogenesis, when they stop dividing A subset of NBs becomes

quiescent until the larval stage, when they enlarge and divide to generate the adult

nervous system Each NB produces a stereotyped lineage of motor neurons, inter

neurons and glial cells and can be identified by its unique position within the

neuroectoderm and its characteristic gene expression profile (Doe, 1992;

Campos-Ortega, 1997; Schmidt et al., 1997) In larval NBs of the central brain, the basic

machinery involved in ACD appears to be conserved with embryonic neuroblasts

However, unlike embryonic NBs, larval NBs divide without a fixed orientation

1.2.1.1 Establishment and maintenance of NB apicobasal polarity

The polarity of NBs is first established by the Par complex, an evolutionarily

conserved protein complex consisting of three proteins: Bazooka (Baz; Drosophila

homologue of C elegans PAR-3), Par-6, and atypical protein kinase C (aPKC) The

Par complex is expressed in the neuroectoderm and localizes as a crescent at the

apical cell cortex in the delaminated NB (Kuchinke et al., 1998; Petronczki and

Knoblich, 2001; Schober et al., 1999, Wodarz et al., 2000) During NB delamination

the protein Inscuteable (Insc) becomes expressed (Kraut et al., 1996) Insc binds the

Par-6/Baz/aPKC complex through Baz, and recruits the GoLoco-motif protein Partner

of Inscuteable (Pins), which recruits the heterotrimeric G protein subunit Gαi

(Schoeber et al., 1999; Parmentier et al., 2000; Schaefer et al., 2000; Wodarz et al.,

2000; Yu et al., 2000; Schaefer et al., 2001; Yu et al., 2005) and coiled-coil protein

Mushroom body defective (Mud) (Bowman et al., 2006; Izumi et al., 2006; Siller et

al., 2006) These two apically-enriched cortical Baz/Par-6/aPKC and Insc/Pins/Gαi/Mud complexes are responsible for apicobasal polarity in the NB and

are inherited by the daughter cell that retains NB identity (Wodarz et al., 1999;

Petronczki and Knoblich 2001; Rolls et al., 2003)

The Rho GTPase Cdc-42 is required to anchor Par-6 and aPKC at the apical

cortex of NBs In cdc42 mutants the localization of Par-6 and aPKC fails and the

overexpression of Cdc-42 leads to ectopic Par-6-aPKC localization However, Baz

localization is not altered in cdc42 mutants Hence, Cdc-42 acts downstream of Baz to

regulate the localization of Par-6 and aPKC (Atwood et al., 2007)

It has been shown that maintenance of the apical localisation of the Insc/Par

complex through subsequent mitotic cell cycles requires contact between NBs and

neuroectodermal cells (Siegrist and Doe, 2006) Isolated and cultured NBs fail to

maintain the position of the Insc/Par complex and spindle orientation This suggests

that extrinsic cues from the overlying epithelium provide positional information for

correct tissue polarity and alignment of the mitotic spindle The identity of extrinsic

cues from the overlying epithelium is still unclear but possible candidates are signalling molecules such as extracellular matrix proteins expressed in neuroectodermal cells (Siegrist and Doe, 2006)

1.2.1.2 Coordination of mitotic spindle orientation with cortical polarity

To ensure the exclusive segregation of cell-fate determinants to the GMC

daughter, the NB mitotic spindle has to be oriented orthogonal to the apical protein

complexes (Kraut et al., 1996; Kaltschmidt et al., 2000) Pins is recruited to the apical

cortex through its interaction with Insc Binding of Gαi facilitates Pins apical cortical

association (Schaefer et al., 2001) and leads to cooperative binding of Pins to Mud,

the Drosophila orthologue of NuMa, a vertebrate microtubule and dynein-binding

protein (Bowman et al., 2006; Izumi et al., 2006; Siller et al., 2006) Mud can in turn

capture astral microtubules from one of the spindle poles and orient the mitotic

spindle Consistently, mutations in pins, Gαi and mud cause defects in spindle

orientation (Bowman et al., 2006; Izumi et al., 2006; Siller et al., 2006)

Pins and Gαi form a complex in vivo with cortical Discs large (Dlg) protein

(Siegrist and Doe, 2005) In turn, Dlg binds to the Kinesin heavy chain 73 (Khc-73)

which is localised at the plus ends of astral microtubules In this way, Dlg connects

microtubules to the cortical Pins-Gαi complex that must be polarized over one spindle

pole in the dividing cell (Siegrist and Doe, 2005) In dlg/khc-73 mutants, Pins/Gαi can

still bind the apically localised Insc/Par complex, but the spindle pole fails to connect

to cortical polarity cues, similarly leading to misoriented mitotic spindles (Siegrist

and Doe, 2005)

1.2.1.3 Spindle asymmetry and differential cell size

In NB divisions, the mitotic spindle is initially symmetric, with the metaphase

plate placed at the centre of the cell At anaphase, the apical aster enlarges and the

basal aster shrinks as spindle microtubules elongate at the apical side and shorten at

the basal side, generating an asymmetric spindle This results in a shift of the

cleavage plane and the generation of two daughter cells of different size (Bonaccorsi

et al., 2000; Kaltschmidt et al., 2000)

The Baz/Par-6/aPKC and Pins/Gαi complexes act in a redundant manner to

regulate NB spindle asymmetry and cell size Single mutations in either complex do

not cause substantial cell size or spindle symmetry defects However, double mutants

affecting both complexes result in symmetric NB divisions (Cai et al., 2003; Fuse et

al., 2003; Yu et al., 2003; Izumi et al., 2004)

The heterotrimeric G-proteins Gβ13F and Gγ1 also play a crucial role in the

regulation of NB spindle geometry and cell size asymmetry (Fuse et al., 2003; Yu et

al., 2003; Izumi et al., 2004) Via a receptor-independent heterotrimeric G-protein

cycle (Bellaiche and Gotta, 2005; Wodarz, 2005), binding of the GDP dissociation

inhibitors Pins and Locomotion defective (Loco) to Gαi promotes the dissociation of

Gαi and maintains it in a GDP-bound state (Wodarz, 2005), leading to the release of

Gβγ (Yu et al., 2000; Schaefer et al., 2001) In Gβ13F null mutants, NB spindle

asymmetry is lost, leading to equal-sized daughter cells with high penetrance In

contrast, overexpression of Gβ13F leads to a reduction in spindle size (Fuse et al,

2003) Gγ1 mutants reveal a phenotype that is almost identical to Gβ13F mutants

(Izumi et al., 2004) Double mutants of Loco and Pins exhibit similar phenotypes to

Gβ13F and Gγ1 mutants (Yu et al., 2005)

1.2.1.4 Centrosome asymmetry

Combined live imaging of spindle assembly and centrosome function in larval

brain NBs (Rebollo et al., 2007; Rusan and Peifer, 2007) has shown that both

centrosomes display asymmetric behavior during NB division After cytokinesis, both

centrosomes are initially located at the region where apical markers were last localized Soonafter, one centrosome stays fixed at the apical cortex organizing an

aster that will be the main microtubule network during most of the NB interphase

Orientation of the future mitotic spindle can be accurately predicted from the position

of this apical aster The second centrosome, which has little, if any, pericentriolar

material (PCM) and does not display any significant microtubule organisation activity, moves extensively throughout the cytoplasm, and slows down near the basal

cortex shortly before mitosis, recruits PCM and organizes the second mitotic aster

Thus, the two centrosomes of an NB are very different structurally and functionally

They are also unequal in fate as the apical centrosome remains in the stem-cell like

daughter cell, whereas the other centrosome goes into the differentiating daughter

(Rebollo et al., 2007; Rusan and Peifer, 2007)

1.2.1.5 Mechanisms of segregating cell fate determinants

The differential fate of the NB daughters is because of the asymmetric localization and preferential segregation of cell-fate determinants such as Prospero

(Pros), Brain tumour (Brat) and Numb to the basal cortex, to be exclusively inherited

by the GMC daughter

Asymmetric segregation of the pros mRNA adaptor Staufen, Pros, and Brat

follows from their interaction with the coiled-coil adaptor protein Miranda (Mira)

(Ikeshima-Kataoka et al.; 1997, Shen et al., 1997; Schudlt, 1998,) In mira mutants,

all three determinants are uniformly cytoplasmic and segregate equally into both

daughter cells Live imaging indicates that the adaptor protein for Numb, Partner of

Numb (Pon) is recruited to the cortex of the mother cell upon entry into mitosis and

then becomes enriched on the basal side (Lu et al., 1998) Quantitative analysis shows

that GFP-Pon and Numb-GFP readily exchange between the cytoplasm and the cell

cortex, which suggests that basal enrichment of Pon and Numb is a result of their

preferential recruitment from the cytoplasm to the basal cortex (Mayer et al., 2005)

Basal enrichment of Pon and Mira occurs in response to the apical–basal

polarity that has been established by the apically-enriched cortical Baz/Par-6/aPKC

and Insc/Pins/Gαi complexes Mutations in baz, insc, pins or mud result in defective

Pon and Mira distribution during metaphase However these defects are often rescued

by telophase (Schoeber et al., 1999; Wodarz et al., 1999; Peng et al., 2000) This

correction of asymmetric basal protein localization defects during late stages of

mitosis is referred to as “telophase rescue” (Peng et al., 2000; Cai et al., 2001)

Telophase rescue is mediated by an interaction of the mitotic spindle with the

overlying cell cortex (Siegrist and Doe, 2005) The Pins/Dlg/Khc-73 complex functions during late mitosis to reorient cortical polarity and rescue determinant

segregation in many (but not all) NBs, in spite of the presence of misoriented

spindles Again, G protein signalling may be involved in telophase rescue, as overexpression of delocalized, truncated versions of Pins and overexpression of Gαi

leads to mislocalization of cell fate determinants (Schaefer et al., 2001; Yu et al.,

2002) Further, simultaneous removal of members of the Snail family Zn-finger

proteins, Snail (Sna), Escargot (Esg) and Worniu (Wor) down-regulates telophase

rescue, suggesting that genes essential for telophase rescue are regulated positively by

the Zn-finger proteins (Ashraf and Ip, 2001; Cai et al., 2001) Interestingly, when Insc

is expressed ectopically in NBs deficient for sna/esg/wor, wild-type asymmetric

divisions are restored These observations suggest that telophase rescue is a cryptic

process that is only needed when apical complex function is disrupted

The segregation of cell fate determinants also involves the interaction of the

Par complex with the cortically localized tumour suppressor proteins Lethal (2) giant

larvae (Lgl), Dlg, and Scribbled (Scrib) (Peng et al., 2000 ; Betschinger et al., 2003)

Phosphorylation of Lgl by aPKC results in its dissociation from the apical cortex

(Betschinger et al., 2003) Hence, Lgl is only active at the basal cortex where aPKC is

absent In lgl mutants, apical polarity is normal, but Pon/Numb localize uniformly at

the cell cortex and in the cytoplasm, whereas Mira/Pros are found uniformly cortical

as well as at the mitotic spindle (Ohshiro et al., 2000; Peng et al., 2000) Accordingly,

overexpressed non-phosphorylatable Lgl (Lgl-3A) is present exclusively at the cell

cortex Conversely, overexpression of a mutant aPKC that is expressed cortically

displaces Lgl from the cortex; as a result, Mira is present throughout the cytoplasm

and is inherited by both daughter cells (Betschinger et al., 2003) In addition, aPKC

has been shown to phosphorylate Numb, hence releasing it from the apical NB cortex

(Smith et al., 2007)

Functional relationships have been reported between Lgl and actin-dependent

non-muscle myosins, such as myosin II (Myo II) and myosin VI (Drosophila Zipper

and Jaguar respectively) These myosins are required to transport cell-fate

determinants and their adaptor proteins to the basal pole of the NB by a process

dependent on the actin cytoskeleton (Barros et al., 2003; Petritsch et al., 2003) At the

basal cortex, active Lgl binds to (Strand et al., 1994) and inhibits myosin II As Lgl is

phosphorylated and inhibited by aPKC at the apical cortex of metaphase NBs (Betschinger et al., 2003), myosin II is active in this apical domain The apically

restricted myosin II modifies the actin cytoskeleton at the apical cortex, preventing

the binding of Mira/Pros and Pon/Numb there and thus, restricting them to the basal

cortex (Knoblich et al., 1995; Lu et al., 1998; Barros et al., 2003) In myosin II

mutants, the basal components of NBs no longer localize asymmetrically and adopt a

cytoplasmic distribution or associate with spindle microtubules Myosin VI has been

shown to bind Mira directly and to transport it to the basal pole of metaphase NB

(Petritsch et al., 2003) Indeed, both myosin VI and myosin II co-immunoprecipitate

with Mira in vivo These studies link the requirement of Lgl with the basal

localization of cell-fate determinants through the action of non-muscle myosins

Vesicle transport and exocytosis may also be involved in basal protein targeting since the yeast homologues of Lgl, Sro7 and Sro77, associate with the

t-SNARE Sec-9 and help target vesicles to the plasma membrane (Lehman et al.,

1999) Therefore, Lgl might similarly promote the delivery of Pon and Mira to the

cell membrane in D melanogaster neuroblasts

1.2.1.6 Cell cycle regulators and asymmetric protein localization

Asymmetric localization of proteins is linked to cell cycle progression The

cell cycle regulator Cdc2 functions in the embryonic CNS by maintaining the correct

localization of the apical protein Insc, though Cdc2 is not necessary for Insc initial

establishment (Tio et al., 2001) Only high thresholds of Cdc2 activity allow a normal

asymmetric cell division through a tight temporal regulation of Insc localization

In the Drosophila larval CNS, the cell cycle regulator Aurora A kinase

(Aur-A) regulates the localization of Numb (Lee et al., 2006, Wang et al., 2006) Aur-A

prevents aPKC localization at the basal pole As aPKC negatively regulates Numb

localization, the inhibition of aPKC by Aur-A allows the accumulation of Numb in

the basal pole In aur-A mutants, Numb localization is deregulated and an increase in

larval NBs is observed (Lee et al., 2006, Wang et al., 2006)

Numb localization may also be regulated by the phosphorylation of its adaptor

protein Pon by another cell cycle regulator Polo kinase (Wang et al., 2007) Pon is not

phosphorylated in polo mutants and, as a result, its partner Numb is not segregated to

the basal cortex of metaphase NBs and is symmetrically distributed cortically

Moreover, the apical protein aPKC is also uniformly distributed in the cortex of polo

mutant NBs Hence, the symmetric distribution of aPKC to both daughter cells,

together with insufficient amount of Numb to inhibit Notch in either daughter cell,

can explain the extensive proliferation in polo mutations (Wang et al., 2007)

The mitotic regulator anaphase-promoting complex/cyclosome (APC/C) also

functions during asymmetric cell division to basally locate the adaptor protein Mira

and its cargo proteins, the cell fate determinants Pros and Brat in the mitotic NB

Mutations in different APC/C core subunits lead to a reduction of basal Mira and to

an increase in its cytosolic accumulation It has been proposed that the reduction in

Mira basal localization observed in APC/C mutants may be due to a loss of Mira

ubiquitylation by APC/C (Slack et al., 2007)

1.2.1.7 Role of cell fate determinants

Several asymmetrically segregating cell fate determinants have been identified

for specifying GMC fate in the larval brain The phosphotyrosine-binding

domain-containing protein Numb can bind the intracellular domain of Notch and repress

Notch signalling (Schweisguth, 2004; Le Borgne et al., 2005) Notch is necessary and

sufficient for promoting larval NB proliferation and suppressing neuronal differentiation Numb is segregated to to the future GMC where it acts to antagonize

Notch Conversely, Notch is asymmetrically activated in the NB daughter where it

acts to promote self-renewal and suppress differentiation (Lee et al., 2006; Wang et

al., 2006) Loss of function mutants affecting Numb lead to aberrant GMC specification (Lee et al., 2006; Wang et al., 2006)

A second determinant is the homeodomain transcription factor Pros pros

mutants fail to express many GMC-specific markers and exhibit axonal defects (Doe

et al., 1991; Vaessin, 1991) Pros can act both as a transcriptional activator and

repressor to induce neuronal differentiation genes and repress neuroblast identity

genes (Mira, Wor and Deadpan), as well as genes which promote cell cycle

progression (string, cyclin A, cyclin E and E2F) (Knoblich et al., 1995; Spana and

Doe, 1995; Choksi et al., 2006)

Brat, a member of the evolutionarily conserved NHL (Ncl-1, HT2A and

Lin41) domain family, is another cell fate determinant inherited by the GMC (Bello et

al., 2006; Betschinger et al., 2006, Lee et al., 2006) Brat acts as a translational

repressor with dozens of possible targets, which is required to restrain cell growth

(Sonoda and Wharton, 2001; Frank et al., 2002; Loop et al., 2004) Brat may regulate

proliferation by modulating the transcription factor Myc (Betschinger et al., 2006)

Fate transformations are limited in brat mutant embryos, but are dramatic in brat pros

double mutant embryos This indicates that Pros and Brat have partially redundant

roles in embryonic neuronal cell-fate specification (Bello et al., 2006; Betschinger et

al, 2006)

1.2.1.8 Asymmetric cell division and tumour suppression

Transplantation experiments have dramatically demonstrated that NB asymmetric division machinery is linked to the control of proliferation and tumour

formation (Caussinus and Gonzalez, 2005) Transplanted tissue from larval brain

containing neuroblasts mutant for the cell fate determinants and their adaptor proteins

overproliferate and form tumours display metasthetic behaviour and kill the host In

pros or numb mutants, GMCs adopt a neuroblast-like fate, resulting in massive

overproliferation (Lee et al., 2006; Wang et al., 2006) Larval brain tissue mutant for

brat develop massive malignant tumours in allograft culture (Caussinus and

Gonzalez, 2005) and displays significant overgrowth in situ (Bello et al., 2006,

Betschinger et al., 2006, Choksi et al., 2006, Lee et al., 2006) Consistently, Mira loss

of function also leads to overgrowth in situ (Betschinger et al., 2006) and malignant

tumour growth in allograft cultures (Caussinus and Gonzalez, 2005) lgl mutant

neuroblasts also exhibit an overproliferation phenotype (Lee et al., 2006)

Some elements of the apical cortex complexes can also, if disrupted, cause

overgrowth Ectopic cortical localization of aPKC inhibits cortical localization of

basal protein complexes (Betschinger et al., 2003; Rolls et al., 2003; Smith et al.;

2007), and leads to a dramatic increase in NB numbers in situ (Lee et al., 2006) pins

mutant NBs have been observed to divide symmetrically by live imaging (Rebollo et

al., 2007), and malignant tumours develop from pins mutant tissue in allograft culture

(Caussinus and Gonzalez, 2005) Spindle misorientation defects in mud mutant NBs

can also cause overgrowth (Bowman et al., 2006; Izumi et al., 2006; Siller et al.,

2006)

Finally, some of the cell cycle regulators also function as tumour suppressors

Larval brain mutants for aur-A or polo have supernumerary NBs (Wang et al., 2006;

Lee et al., 2006; Wang et al., 2007) and develop as malignant tumours when

implanted in adult hosts (Castellanos et al., 2008)

Figure 2 ACD in the Drosophila NB The components of the apical complex interact

with each other to establish polarity, position the spindle and localize cell fate

determinants to the basal daughter cell The cell-fate determinants and their adaptors

are inherited by the basal cell The figure is adapted from Zhong and Chia (2008)

1.2.2 Drosophila Sensory Organ Precursors (SOP)

SOP cells delaminate from a polarized epithelium and generate the

Drosophila external sensory organs through a series of stereotypic asymmetric

divisions (Figure 3) Asymmetric division of the SOP pI cell yields two secondary

precursor cells: the anterior cell, pIIb, and the posterior cell, pIIa pIIb gives rise to a

neuron and a sheath cell, whereas pIIa gives rise to a socket cell and a hair cell PNS

precursors divide within the plane of the epidermis with the mitotic spindle orientated

along the anterior-posterior (A-P) axis

1.2.2.1 Establishment of SOP planar polarity and Numb segregation

The Frizzled (Fz) signaling pathway is responsible to polarize the SOP along

the A-P axis and consequently, to orientate the mitotic spindle along this axis (Gho

and Schweisguth, 1998; Bellaiche et al., 2001) The asymmetric distribution of different components of Fz pathway, such as the Dishevelled (Dsh), the four-pass

transmembrane protein Strabismus (Stbm) and the LIM and PET-domain protein

Prickled (Pk) is the key to the establishment of planar polarization

The Fz receptor and its most proximal effector Dsh locate at the posterior pole

of the precursor whereas the other two components of the pathway Stbm and Pk

locate at the anterior pole (Bellaiche et al., 2004) Stbm binds the PDZ protein Discs

large (Dlg) facilitating its anterior localization (Bellaiche et al., 2004) Dlg in turn,

binds Pins (Bellaiche, 2001; Bellaiche et al., 2004) Both proteins Dlg and Pins form

part of the anterior signaling complex Dlg-Pins-Gαi required to control the anterior

localization of cell-fate determinants, such as Numb and its adaptor protein Pon

(Bellaiche, 2001) In addition, Fz cooperates with Dlg and Pins to localize Baz to the

posterior cortex of the precursor (Bellaiche et al., 2001) and both the posterior

Baz-Par-6-aPKC (Bellaiche et al., 2001; Schaefer et al., 2001) and anterior Dlg-Pins-Gαi

complexes (Bellaiche, 2001; Roigiers et al., 2001; Bellaiche et al., 2004) regulate the

asymmetric localization of Pon/Numb to the anterior cortex of the SOP

Other modulators of Numb asymmetric localization include Flamingo (Fmi), a

seven-transmembrane cell-adhesion molecule, which acts downstream of Fz and is

the only member of the Fz pathway whose localization in SOPs is not polarized (Lu et

al., 2000) Fmi mostly locates to the cell-cell boundaries between the precursor and

the surrounding cells In fmi mutants, the localization of Pon and are altered (Lu et al.,

2000) Cell cycle regulators such as Aurora-A and Cdc2 kinases (Berdnik and Knoblich, 2002; Hutterer et al., 2006; Chia et al., 2008) have also been implicated in

Numb asymmetric localization

1.2.2.2 Directional signaling and cell fate difference

The fate difference between pIIb and pIIa originates from directional signalling between the two cells Numb colocalizes during mitosis with Pins-Gαι at

the anterior cortex of SOPs and thus partitions into pIIb (Bellaiche, 2001; Schaefer et

al., 2001) In numb mutants, two pIIa-like cells are generated, whereas overexpression

of Numb yields two pIIb-like cells (Rhyu et al., 1994) Like Numb, the E3 ubiquitin

ligase Neuralized (Neur) localizes during mitosis at the anterior cortex of SOPs and is

thus inherited exclusively by pIIb (Le Borgne and Schweisguth, 2003) In neur

mutants, two pIIb-like cells are generated, which indicates that Neur is needed for

pIIa fate acquisition Both Numb and Neur are regulators of Notch signaling Numb

antagonizes Notch signaling through a direct protein-protein interaction in the daughter cell in which Numb is asymmetrically segregated, both in the PNS and in

the CNS (Guo et al., 1996, Spana and Doe, 1996)

In the pIIb cell, Numb associates with α-adaptin, a component of the AP-2

complex that targets transmembrane proteins for endocytosis (Berdnik et al., 2002)

α-adaptin alleles that encode proteins that are unable to bind Numb exhibit

phenotypes that are similar to those of numb mutants, which suggests that α-adaptin is

crucial for downregulating Notch activity in pIIb (Berdnik et al., 2002) The

trans-membrane protein Sanpodo, which associates with Numb and is a positive regulator

for Notch signalling (Skeath and Doe, 1998), has been suggested to be a target of

α-adaptin-mediated endocytosis (Berdnik et al., 2002; Hutterer and Knoblich, 2005)

Indeed, whereas Sanpodo is present at the cell membrane of pIIa and localizes on

endosomes in pIIb in the wild-type, it is found at the plasma membrane of both pIIa

and pIIb in cells that lack Numb or α-adaptin (Hutterer and Knoblich, 2005)

However, blocking endocytosis does not prevent Notch signalling in Drosophila

(Seugnet et al., 1997), and numb mutants that lack domains that are essential for

binding with endocytic proteins, including α-adaptin, can nevertheless specify cell

fate in the nervous system (Tang, 2005) Thus, Numb can specify cell fate independently of endocytosis

The E3 ubiquitin ligase Neur present in the pIIb daughter cell ubiquitinates the

Notch ligand Delta and promotes its endocytosis (Lai et al., 2001; Pavlopoulos, 2001,

Yeh et al., 2001, Le Borgne and Schweisguth, 2003) Delta is secreted from pIIb and

activates the Notch receptor on pIIa, thus regulating two transcriptional programmes

that are important for fate specification The first programme entails the transcriptional repressor Tramtrack p69 (TTK69), which is present only in pIIa (Guo

et al., 1995; Okabe et al., 2001) ttk69 mRNA is present in both pIIb and pIIa, but its

translation is prevented in pIIb by the RNA-binding protein Musashi; Notch signalling prevents Musashi-dependent translational repression in pIIa (Okabe et al.,

2001) In the second programme, the processed form of Notch that is present in pIIa

associates with Suppressor of Hairless to form a transcriptional activator complex

strictly in this cell (Schweisguth and Posakony, 1994, Zeng et al., 1998)

Directional signaling

Figure 3 ACD in the Drosophila SOP cell The Par complex and the Pins-Gαi

complex are polarized to opposite poles of the SOP pI cell, leading unequal segregation of cell fate determinants to one of the daughter cells The figure is

adapted from Gonczy (2008)

1.2.3 Drosophila Germline Stem Cells

Drosophila germline stem cells (GSCs) are totally dependent on direct contact

with a “niche” of specialized cells for asymmetric cell division GSCs that abandon

their niche lose SC identity and differentiating daughter cells that establish ectopic

contact with the niche might acquire SC identity (Brawley and Matunis, 2004, Kai

and Spradling, 2004) Cells that form the “niche” of the germline regulate GSC

self-renewal via short range diffusible signals

In the female ovary, cap cells form the GSC niche SCs and cap cells are

connected by adherens junctions that contain both β-catenin and DE-cadherin Removal of either one of these proteins from the GSCs results in stem cell loss

suggesting that niche adhesion is essential for stem cell maintenance (Song et al.,

2002) Cap cells synthesize the ligands called Decapentaplegic (Dpp) and Glass

bottom boat (Gbb) that activate Bone morphogenetic protein (BMP) signalling in

germline stem cells, thereby repressing the gene bag of marbles (Chen and McKearin,

2003; Song et al., 2004), which encodes a protein that promotes differentiation

(Ohlstein and McKearin, 1997) Upon division of the stem cell, one of the two

daughter cells loses direct contact with the niche, receives an attenuated BMP signal

and initiates Bam transcription Bam initiates a characteristic differentiation program

in the cystoblast to form the oocyte and support cells (Fuller and Spradling, 2007)

Male GSCs (mGSC) show a similar dependence on the niche for maintenance

of SC identity during asymmetric division (Figure 4) mGSCs within the testis divide

asymmetrically to generate two daughters of equal size One daughter cell stays close

to the niche formed by hub cells, and retains mGSC identity, and the other, the

gonialblast, differentiates to produce a cyst of spermatocytes Hub cells synthesize a

ligand called Unpaired that activates the Jak–Stat (Janus kinase and signal transducer

and activator of transcription) signalling pathway in germline stem cells to prevent

differentiation, presumably by controlling target genes that remain to be identified

(Kiger et al., 2001; Tulina and Matunis, 2001; Yamashita et al., 2005)

Accordingly, mitosis is oriented so that daughter cells are located proximal

(the stem cell) and distal (the differentiating cell) to the hub A specialized region of

the mGSC cortex that contains adherens junctions and is enriched for Adenomatous

polyposis coli protein homologue (APC2), E-cadherin and Armadillo (ARM) seems

to provide a polarity cue or anchor for astral microtubules that emanate from the

centrosome (Yamashita et al., 2003) Interaction of astral microtubules with this

region of the cortex, a process that is mediated by APC2, contributes to controlled

spindle alignment during mGSC division (Yamashita et al., 2003; Li and Xie, 2005;

Yamashita et al., 2005)

In mGSCs, the mother centrosome is always positioned at the interface of the

GSC and the hub cells Upon division the mother centrosome is retained in the GSC

while the newly formed centrosome is inherited by the gonialblast (Yamashita et al.,

2007) The mother centrosome has more PCM than the daughter centrosome In

mutants for Centrosomin (Cnn), which is required for microtubule nucleation and

centrosome anchoring to astral microtubules, the position of mother and daughter

centrosomes is randomised in male GSCs The phenotype is accompanied by an

increase in the number of GSCs This may be the consequence of spindle misorientation leading to the loss of the stereotyped asymmetric division observed in

the wildtype (Yamashita et al., 2007) Alternatively mother and daughter centrosomes

may carry fate determinants themselves and so influence different cell fates in a more

direct way

Figure 4 ACD in the Drosophila mGSC Polarity cues from contact with the Hub

cells allow the dividing mGSC to properly align the spindle and correctly specify the

fates of the two daughter cells The figure is adapted from Gonzalez (2007)

1.3 ASYMMETRIC CELL DIVISION IN VERTEBRATES

Most of the current knowledge about asymmetric cell division in vertebrates is

derived from embryonic progenitor cells in the developing brain Asymmetric cell

division has an important role in other organs in vertebrates, including the hematopoietic system, skin, kidney and muscle (Kiel et al., 2005; Lechler and Fuchs,

2005; Fischer et al., 2006; Cossu and Tajbakhsh, 2007)

1.3.1 Neural progenitor cells

Brain progenitors reside in a polarized environment and are thought to use this

polarity to generate identical copies of themselves but also other cells that differentiate into neurons Radial glial cells (RGCs) are a major group of neural

progenitor cell within the ventricular zone (VZ) RGCs can divide symmetrically,

generating two progenitors (“proliferative” divisions), or asymmetrically, generating

one progenitor and a differentiating neuron (“neurogenic” divisions) (Chenn and

McConnell, 1995; Cayouette and Raff, 2003; Haydar et al., 2003, Noctor et al., 2004)

Symmetric divisions are prevalent during early neurogenesis, increasing the

pro-genitor pool size, whereas asymmetric divisions become more frequent thereafter,

allowing more differentiated cells to be produced (Konno et al., 2008; Noctor et al.,

2008)

1.3.1.1 Mitotic spindle orientation and modes of cell division

It has been suggested that vertical divisions, with mitotic spindle roughly

aligned along the ventricular surface (planar polarity) are symmetric, whereas horizontal divisions, with the spindle aligned along the apical-basal axis are asymmetric (Figure 5) (Chenn and McConnell, 1995; Cayouette and Raff, 2003;

Haydar et al., 2003, Noctor et al., 2004; Konno et al., 2008) However, this is not a

general rule, as recent findings in the mouse forebrain indicate that a parallel spindle

orientation usually yields an asymmetric division (Konno et al., 2008)

The mammalian counterparts of Drosophila Insc and Pins, mInsc, and AGS-3

and LGN respectively, are required for mitotic spindle orientation along the

apical-basal axis (Sanada and Tsai, 2005; Zigman et al., 2005) mInsc is asymmetrically

localized to the apical side of VZ progenitor cells (Zigman et al., 2005) Increased

mInsc expression correlates with the switch from symmetric to asymmetric division

during neurogenesis In contrast, RNAi knockdown of mInsc inhibits asymmetric

division and favors symmetric division (Zigman et al., 2005) AGS-3, a non-receptor

activator of Gβγ subunits of heterotrimeric G proteins, is required to trigger Gβγ

signaling in cerebral cortical progenitors When G-proteins are inhibited or when

AGS-3 is targeted by RNAi in RGCs, the number of symmetric divisions increases at

the expense of asymmetric divisions (Sanada and Tsai, 2005)

Par-6 and the vertebrate homologues of Par-3 and aPKC, ASIP, PKCξ and

PKCλ, respectively localize apically and are concentrated in adherens junctions (Manabe et al., 2002) Overexpression of Par-6 selectively increases the number of

symmetric divisions without affecting the number of asymmetric divisions (Costa et

al., 2008)

1.3.1.2 Segregating cell fate determinants

No segregating determinant that has been found in flies has been found to act

as a segregating determinant in the mammalian brain Numb is basolaterally localized

in mitotic radial glia cells and in vesicles near adherens junctions of the apical-end

feet of interphase RGCs surrounding the dividing progenitors (Rasin et al., 2007)

Numb and the Numb-related protein numblike (Nbl) mutant mice exhibit premature

depletion of neural progenitor cells (Peterson et al., 2002; 2004) and defects in cell

polarity (Rasin et al., 2007) However, adherens junctions are lost from radial glia

cells in the absence of numb and this may explain the morphological defects observed

in the mutant mice Thus, Numb regulates epithelial polarity but might not actually be

a segregating determinant in mouse neural progenitor cells

Nbl is present throughout the cytoplasm (Zhong et al., 1997) and does not

show any asymmetric segregation described for Numb The Pros homologue, Prospero-related homeobox 1 (Prox1) does not seem to segregate asymmetrically

(Dyer et al., 2003) The Brat homologs, Tripartite motif (TRIM) proteins have

non-ubiquitous, differential expression patterns in the embryonic nervous system (Reymond et al., 2001), suggesting diverse roles in neural development More specifically, TRIM-3 has a role in neurite outgrowth in PC-12 cells (El-Husseini and

Vincent, 1999; El-Husseini et al., 2000; Reymond et al., 2001), and its close sequence

relative TRIM-2 may be involved in activity-dependent neuronal plasticity (Ohkawa

et al., 2001) In a recent study, TRIM-32 is shown to become polarized in dividing

progenitors and be concentrated in one of the two daughter cells TRIM-32 overexpression induces neuronal differentiation while inhibition of TRIM-32 causes

both daughter cells to retain progenitor cell fate (Schwamborn et al., 2009)

However, the EGF receptor (EGFR) shows a polarized distribution in dividing

mouse neural progenitors (Sun et al., 2005) and is sometimes preferentially inherited

by one of the two daughter cells However, this asymmetric segregation is only seen

in a fraction of the progenitors and can by no means explain all asymmetric divisions

Furthermore, EGFR also localizes asymmetrically in the subventricular zone where

cells are supposed to divide symmetrically In cell culture, however, the daughter cell