master thesis final 8 -...

Understanding the Role of the Actin Cytoskeleton in Vesicle Trafficking to Restrict Growth of Drosophila Epithelia

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I. Introduction

The ability of eukaryotic cells to assume a variety of shapes and to carry out

coordinated and directed movements depends on a complex network of protein

filaments that extends throughout the cytoplasm. The extent and pattern of actin

filament polymerization drives the protrusive force and shape of migrating cells, as

well as the cortical tension necessary for maintaining adhesion contacts between cells

and between the cell and its substrate.

1. Actin dynamics and its regulation by actin binding proteins

Actin exists in a cell in two main forms: the monomeric globular actin (G‐actin),

and the polymeric filamentous actin (F‐actin). Under physiological conditions, ATP

bound G‐actin incorporates into growing filaments at a fast‐growing barbed (+) end,

undergoing thereafter a slow hydrolysis into ADP‐actin, as actin monomers are shifted

along the filament towards the slow‐growing pointed (‐) end 1. Actin dynamics and

structure are controlled by a large variety of Actin Binding Proteins (ABPs), including

actin nucleators, depolymerisation factors, actin‐bundling proteins and actin‐

crosslinking proteins. Furthermore, some ABPs link filaments to the plasma membrane

or organelles within the cell, while others use actin filaments as tracks upon which to

move vesicles and organelles 2‐4.

1.1 Actin filament nucleation

The nucleation of actin filaments is an energetically unfavourable event, being

promoted by certain ABPs, such as the Arp2/3 complex and formins. For instance, the

Arp2/3 complex can generate a stable trimer with G‐actin along the side of an actin

filament, producing a new filament branch 5 (Fig.1).

Extensive nucleation occurs mainly at the leading edge of motile cells, where

the meshwork of filaments acts as a platform against which actin polymerization can

push 6, 7 (Fig.x). The morphology of the membrane protrusion varies according to the

growth extent and bundling of the filaments, ranging from filopodia, that arise from

long and bundled filaments, to lamellipodia, that rely on a highly branched network of

Gaspar P. Master Thesis

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short actin filaments. In general, actin polymerization induced by the Rho‐like GTPases

Cdc42 and Rac, respectively promotes the formation of filopodia and lamellipodia,

downstream of the Arp2/3 complex 5.

Fig.1 – Actin dynamics is controlled by a large array of ABPs. The actin nucleators, such as the Arp2/3

complex, promote de novo actin filament nucleation and branching. Profilin sequesters G‐actin

monomers, translocating them to sites of active filament assembly and promoting prolimerization.

ADF/cofilin factors severe the actin filaments and promote dissociation of the actin monomers from the

pointed (‐) end. Capping proteins (CPs) restrict the access to the barbed (+) end, forming a protein cap

that impedes further addition and loss of actin monomers.

1.2 Actin filament depolymerization and sequestration of actin monomers

Actin depolymerization factors also regulate the extent and spatial pattern of

actin polymerization. For instance, members of the ADF/Cofilin family are known to

dramatically accelerate actin filament turnover in vitro 8, which occurs mainly through

filament severing and monomer dissociation from the pointed end 9 (Fig.1). Similar to

the Arp2/3 complex, ADF/Cofilin seems to be regulated by the activity of Rho‐like

GTPases. For instance, Rho and Cdc42 respectively induce the formation of actin stress

fibers and filopodia 10, 11.


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Besides ADF/Cofilin, other ABPs, like the Cyclase Associated Proteins (CAPs),

regulate the availability of actin monomers by sequestering them and prevent their

incorporation into filaments 2, 12. On the other hand, Profilins enhance exchange of

ADP for ATP on sequestered actin monomers, thereby promoting actin filament

polymerization 13. A generally devised model of actin filament ‘tread‐milling’ assumes

that filament turnover is driven by actin monomer dissociation through ADF/Cofilin,

whereas at sites of rapid actin assembly, the pool of ADP‐actin monomers undergoes a

rapid nucleotide exchange driven by Profilin, to promote new incorporation into the

actin filaments 14 (Fig.1).

1.3 Actin filament termination

Actin filament termination occurs mainly through the activity of filament end‐

binding proteins, such as proteins of the Gelsolin superfamily and Capping proteins

(CPs). For instance, gelsolin is able to dissolve actin gels in vitro 15, but it is still

controversial whether it does so through a severing or a capping activity over the

actin filaments 16, 17. On the other hand, CPs form a highly conserved αβ heterodimer

(CP) that binds to the barbed end of actin filaments, thereby forming a protein cap

that arrests actin polymerization by locally preventing the further addition and loss of

actin monomers 18 (Fig.1). Functional CP, along with the Arp2/3 complex, support the

assembly of a dominantly branched network of small actin filaments, giving rise to the

lamellipodia of migrating cells 19(Fig.2). When capping of actin filaments by CP is

inhibited, long actin filaments can become bundled, giving rise to filopodia 20.(Fig.2)

Intriguingly, besides having a critical role in the termination of filaments, CP has been

suggested to mediate actin filament attachment to the plasma membrane 21, 22.

Although much is now understood about how actin dynamics is regulated,

most work addressing this issue has been developed in cultured cell lines. Few studies

have addressed the importance of cytoskeletal dynamics in the regulation of tissue

homeostasis and morphogenesis in the context of an intact metazoan organism 23.

Throughout this thesis I will concentrate on the role of actin dynamics during

epithelial development of the Drosophila wing imaginal disc.


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Fig.2 – Functional CPs and the Arp2/3 complex support the assembly of a dominantly branched

network of small actin filaments, giving rise to lamelipodia. (b) When the capping of actin filaments is

inhibited, actin filaments can become bundled, giving rise to filopodia. Some molecular components

have been identified that could inhibit or counteract the capping activity: the Ena/VASP family of

proteins seems to exhibit anticapping and antibranching activities and PIP2, CARMIL and

Melanotrophin/V‐1 have been proven to bind and block the C‐terminal capping portion of CP, in vitro 24‐27.

2. Epithelial cell‐cell adhesion and polarity

Simple epithelia generally consist of a laterally coherent layer of cells that have

a distinct apico‐basal polarity, having a apical surface that borders a lumen, a lateral

surface that adheres to neighbouring cells, and a basal surface that adheres to the

extracellular matrix (ECM) or basal lamina 28. A great variety of epithelia line the walls

of cavities and channels or, in the case of the epidermis, serve as the outside covering

of the body, pertaining a variety of functions, such as the selective absorption of


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solutes, the glandular production of hormones, digestive enzymes and termo‐

regulatory fluids, and serving as a physical and immunological barrier against pathogen

attack.

Epithelial cells have a polarized actin system, with a band of filaments

concentrated at the apical zonula adherens (ZA), long actin filaments around the

basolateral cortex and stress fibres localized at the basal surface. The apical actin

network supports cell‐cell adhesion through the adherens junctions (AJ), whereas cell‐

matrix adhesion is supported by stress fibre anchoring to hemiadherens junctions 29.

Cell‐cell adhesion is mainly accomplished by cadherin homophilic interaction

between cells at the AJ 30. In turn, the cytoplasmic domain of cadherins is able to

interact with proteins of the armadillo repeat family, such as Armadillo/β‐catenin

(Arm) and p120‐catenin, thereby regulating actin dynamics at adhesive contacts 31.

For instance, the cadherin‐β‐catenin complex is thought to trap α‐catenin, which

would otherwise compete with the Arp2/3 complex in binding to the actin filaments 32, 33. Although by no means clarified, the apical ‘actin belt’ seems to be of great

importance for the establishment and maintenance of cell‐cell adhesion, therefore

assuring proper epithelium development.

Besides the cadherin complexes, other macromolecular complexes are required

for the establishment of cell‐cell adhesion. For instance, in the subapical region (SAR)

of epithelial cells, the Bazooka (Baz/Par3)/Par6/aPKC complex is required for

establishing the AJ, mainly through the recruitment of apical polarity determinants and

the Rho‐like GTPases Cdc42 and Rac 34‐39. In Drosophila epithelial cells, the mature

Par6‐aPKC complex drives the apical recruitment of the transmembrane protein

Crumbs (Crb) and its cytozolic associates, Stardust (Sdt) and Discs Lost/DPatJ (Dlt),

whose activity is required to further recruit and concentrate sparsely distributed DE‐

cadherin into the AJ 40, 41 (Fig.3). Thus, mutants for components of the Crb polarity

complex fail to develop a normal AJ, with DE‐cadherin being diffusely distributed and

with apical and basolateral membrane markers overlapping each other 42, 43.

Functional regions of Crb residing in its cytoplasmic tail assure the correct

biogenesis of the AJ and the establishment of apical‐basal polarity 41, 44. In particular,

the cytosolic tail of Crb interacts with moesin, an ERM protein that tethers the

complex to the actin cytoskeleton 45. Interestingly, in Drosophila, expression of


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cytoplasmic truncated versions of Crb leads to loss of proper DE‐cad distribution and

consequent multilayering of simple epithelia 44.This phenotype seems to be linked to

the ability of the Crb complex to repress the activity of Discs‐large (Dlg), Scribble (Scrib)

and Lethal giant larvae (Lgl) 46, 47(Fig.3). These proteins form the septate junction (SJ) in

Drosophila, being the functional equivalent to the vertebrates’ tight junction, although

being localized below the AJ. Importantly, the SJ constitutes the paracellular barrier

and restricts the localization of apical polarity determinants, such as Crb (Fig.3). When

mutated, the components of the SJ cause epithelial cells of Drosophila to aberrantly

distribute Crb and DE‐Cadherin. As a result, epithelial cells become unpolarized, giving

rise to a multilayered tissue that overproliferates and acquires invasive properties 48, 49

(Fig.3).

Fig. 3 – Epithelial polarity complexes are located in stratified regions along the apico‐basal axis of cells.

In Drosophila, the subapical is located above the zonulae adherens and the septate junction is located

below. The Crb/Sdt/Dlt complex is located in the SAR and, along with the Baz/Par6/aPKC complex, is

responsible for maintaining the integrity of the AJ, counteracting the basolateral Lgl/Scrib/Dlg septate

complex. In Drosophila’s ovarian follicle epithelium, mutant cells for components of the septate junction

overproliferate and stream into the germ line cyst, where they penetrate between nurse cell

membranes 28, 48 (as shown in the series below).


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2.1 Vesicle trafficking and the control of epithelial cell polarity and proliferation

Epithelial cell polarity relies upon polarized transport of proteins and lipids to

the apical, basolateral and junctional domains in a process called exocytosis. In this

case, recently synthesized proteins are released from the trans‐Golgi network (TGN) to

apical or basolateral membrane domains, following specific guiding signals 50. In

addition, many membrane‐linked receptors are recruited into endocytic vesicles being

either recycled back to the plasma membrane or targeted to lysosomal degradation 51,

52 (Fig.4).

Early endosomes, budding from the plasma membrane, are characterized by

their enrichment in certain phosphoinositides and for the presence of Rab5 GTPases.

These endosomes mediate recycling exocytosis to the apical or basolateral membrane

or endocytosis to a degradative lysosomal route. The degradative route is

characterized by a series of maturation steps, leading to progressive endosomal

acidification and to the formation of multivesicular bodies (MVBs). Ultimately, when

the endosome reaches its maturity, Rab7 GTPases mediate its fusion with a lysosome,

where proteolytic degradation occurs 53(Fig.4).

Importantly, the actin cytoskeleton seems to be essential for the sorting of

vesicle cargo to the basolateral membrane, through several sorts of myosin motors 54,

55. For instance, in MDCK cells, the use of actin polymerization inhibitors compromises

the sorting of proteins to the basolateral membrane domains, forcing them to take

apical recycling routes 56. On the other hand, the microtubule network seems to be

essential for transporting vesicle cargo to the apical epithelial surface, essentially

through dynein and kinesin (‐) end‐directed motors 57. A generally devised model is

that, according to the peripheral location of actin filaments in many cell types, the

actin cytoskeleton essentially mediates vesicle trafficking events close to the

membrane, whereas the microtubule network drives ‘long‐range’ transport of vesicles

across the cell cortex 54, 57. Accordingly, for endocytosis to proceed, actin filaments are

known to increase the uptake of membrane‐linked receptors into vesicle‐coated pits,

whereas the microtubule network is required to assure deep‐cortical endocytic

transport and maintain the distribution of the late endocytic compartments 58‐60.


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Fig. 4 – Overview of vesicle trafficking in epithelial cells. The trans‐Golgi network (TGN) releases

recently synthesized proteins to apical or basolateral membrane domains and the recycling endosome,

which mediates recycling exocytosis to the apical or basolateral membrane, through apical recycling

routes. The endocytic pathway can lead to recycling back to the plasma membrane or to lysosomal

degradation, and each route is characterized by endosome compartments that possess typical

regulatory proteins (in red and purple). The endosome markers labelled in purple have been shown to

suppress tumour formation in Drosophila, as indicated by the tumoral phenotypes of the respective

mutant imaginal discs 61 (as shown in the picture to the left).

When endocytosis is impaired, accumulation of proteins occurs at the level of

the plasma membrane or intermediate endosomal compartments. Interestingly,

several tumour suppressor genes (TSGs) have been characterized in Drosophila that

are key components of the endossomal system, being required for endosome

maturation 62.(Fig.4) For instance, genes required for late endosome maturation, such

as Vps23 (erupted, tsg101) and Vps25 have been shown to be neoplastic tumor

suppressors in Drosophila 63‐65. Disrupting the function of these genes seems to

prevent the degradation of membrane signalling receptors, leading to transduction of

an excessive mitogenic or stress signal 66. Interestingly, mutant cells for avalanche, a

synthaxin required for early endossome sorting (Fig.4B), lead to the neoplastic

development of several Drosophila epithelia, which correlates to vesicular

WT

Rab5

avl

vps25

vps23


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accumulation of the polarity determinant Crb 67. However, until now, no evidence has

clarified how endocytosis of this polarity determinant contributes to the regulation of

epithelial growth.

2.2 The development of epithelial neoplasms

During aberrant epithelium development, cells often loose adhesion between

each other, forming epithelial cysts that eventually extrude to the basal lamina.

Frequently, these abnormal tissue configurations disrupt the normal growth of the

tissue, giving rise to morphogenetic distortions and overgrowths typical of epithelial

tumors or neoplasms 68‐70 (Fig.5).

Neoplastic development is a multistep process, which is thought to involve

cooperation between several mutations, as well as between the tumor and its

microenvironment 71. Indeed, in a chimeric tissue environment, abnormal epithelial

cells, that would potentially drive neoplastic transformation, might undergo apoptosis

triggered by their tissue surroundings, a phenomenon designated as cell competition 72, 73. For instance, when a clone of mutant cells for the tumour suppressor scrib arises

among normal tissue, the surrounding wild‐type cells actively eliminate the

unpolarized mutant cells, triggering their apoptosis 74. In this case, a synergistic

interaction with the Ras or Notch oncogenes is required for the formation of scrib

mutant invasive tumors 74, 75. In this sense, multiple alterations, either arising within

the tumor or in the surrounding normal tissue, seem to be required for promoting

unrestrained tumor growth and lethality 76.

In Drosophila, tumoral epithelia have been classified as hyperplastic whenever

they show increased proliferation, although epithelial architecture is maintained, or

neoplastic if tissue overproliferation occurs concurrently with disruption of epithelial

structure, as cells become unpolarized. Neoplastic epithelia are also characterized by

their inability to terminally differentiate, with cells frequently acquiring migratory and

matrix degradation potential, allowing them to cross the basal lamina and invade

adjacent tissues 62, 69 (Fig.5).

During tumour development, loss of cell‐cell adhesion is usually correlated with

disruption of the apical actin network, while metastatic invasion is associated with

enhanced protrusive activity of the basal actin stress fibers 77. In particular, many ABPs


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have been shown to be misregulated during the metastatic process, including the

Arp2/3 complex components, CPs and members of the ADF/cofilin family 78, 79.

Therefore, actin dynamics at adhesive contacts seems to be crucial for assuring proper

epithelial architecture and tissue homeostasis.

Fig.5‐ Monoclonal tumors are thought to arise by the loss of heterozygozity, leading to overexpression

of a certain oncogene or to a decreased expression of a certain tumour suppressor gene. This event is

then thought to trigger complex rearrangements of the cellular architecture, leading to an epithelial‐to‐

mesenchymal transition. Loss of proliferation control and the acquisition of resistance to cell death are

thought to be triggered by this transition. In Drosophila, tumour suppressor pathways have been found

that link cell polarity to proliferation control. In the depicted example, a series of neoplastic epithelia

arise from overexpression of the Crb polarity complex (as show in the A‐C series, compared to WT).

3. Actin dynamics and the control of cell proliferation

The use of drugs to manipulate actin dynamics has been widely used to

investigate links between actin and stress signalling. For instance, several studies

using mammalian cell lines in culture have shown that, depending on the cell type,

either depolymerisation or stabilization of F‐actin is accompanied by the induction of

an apoptotic stress 80. Interestingly, one striking difference between cell types is the

ratio of G‐actin to F‐actin, this being tightly regulated by the activity of several ABPs.

In this sense, several studies have suggested a role of ABPs in regulating cell survival

WT A B C


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and death 80. For instance, gelsolin activity seems to prevent the induction of

apoptosis and ADF/cofilin has been shown to induce cytochrome c leakage and trigger

apoptosis 81‐83. However, very few studies have actually revealed the significance of

ABPs’s function during the homeostatic processes of cell growth and death within

tissues. For instance, recent studies in Drosophila, concerning the role of CP in wing

tissue morphogenesis, may provide us some insight into this question.

3.1 CP prevents cell extrusion within restricted regions of Drosophila epithelia

As expected, in vivo studies showed that CP prevents excessive actin filament

polymerization within Drosophila epithelia, confirming its already known role in actin

filament termination. However, CP doesn’t have the same developmental function in

all epithelia. For instance, loss of either capping protein α (cpa) or β (cpb) subunits in

the wing imaginal discs of Drosophila gives rise to different cellular outcomes along the

proximal‐to‐distal axis 84. The wing imaginal disc of Drosophila can be subdivided into

three fate‐defined regions: the notum, in the most proximal part, then the hinge, and

finally the blade, in the most distal part. In the blade, CP mutant cells lose polarity,

extrude basally and undergo cell death, while mutant cells that develop in the

remainder of the wing disc, sustain polarity and survive 84.(Fig.6) This behaviour is

correlated to a specific actin filament network present in mutant cells: while in the

wing blade epithelium, excessive actin filaments are observed throughout the entire

cell cortex, in the remainder of the wing disc, actin filaments accumulate mainly at the

cell’s apical surface 84.

3.2 CP prevents neoplastic development of Drosophila epithelia

Recently, it was found that cpa mutant cells in the follicle epithelium of

Drosophila are prone to become neoplastic, lacking polarity and cell‐cell adhesion

(Janody, unpublished; Fig.6). Furthermore, RNA interference (RNAi) leading to CP

depletion in the presumptive blade region of the wing imaginal disc, resulted in tissue

overproliferation, possibly due to the outward resistance of depleted cells to cell

death 85 (Fig.6). Interestingly, it was found that like mutations in genes associated with

the endocytic pathway, including the syntaxin avalanche (avl) and Rab5 86, loss of CP

results in punctated Crb accumulation (Janody, unpublished; Fig.6). Overall, the


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similarities between endocytosis defective phenotypes and the ones observed for CP

mutant cells suggest that the actin dynamics machinery is responsible for maintaining

the appropriate network of actin filaments to support vesicle trafficking within

epithelial cells. Interestingly, the Dictyostelium CARMIL protein was found to link CP

and the Arp2/3 complex to Myosin I 87, whose activity seems to involve the transport

of Golgi‐derived vesicles 88. In this sense, CPs might directly be involved in vesicle and

organelle movement or have an indirect role in regulating trafficking events, trough

their effect on actin dynamics.

Fig.6 – CP mutations have different cellular outcomes depending on the tissue context. cpa mutant

clones developed in the hinge or notum are maintained within the wing epithelium, while clones

developed in the wing blade undergo extrusion and apoptosis, as indicated by anti‐activated Caspase3

(C3) staining84 (A). CP mutant clones induced in the ovarian follicle epithelium form a multilayered

epithelium in the anterior and posterior poles of the egg chamber (Janody, unpublished)(B). Extruding

cpa mutant cells (green) accumulate Crb in punctated structures (Janody, unpublished) (C‐C’).

Depletion of CP by RNAi leads to neoplastic overgrowth of the wing epithelium (D‐D’).

B

Arm GFP Dlg/C3

Phal GFP Dapi Crb

Crb GFP

Phal Phal

WT sd>UAScpbRNAiC10

cpa107E clones

cpa69E clones

A

C

C’

D E


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4. Patterning of the Drosophila wing imaginal disc

Highly conserved mechanisms in epithelial development, such as the

establishment and maintenance of epithelial cell polarity, have been intensively

studied in Drosophila, paving the way for the discovery of homologous mechanisms in

vertebrates. In particular, the imaginal discs and the ovarian follicle epithelium of

Drosophila constitute excellent models for the study of epithelial differentiation in

postembryonic development.

The Drosophila egg hatches into a larva that has no resemblance to the adult

fly, since it possesses no external evidence of motile appendages. However, the

primordial adult appendages are already present in the larvae, deriving from

invaginating epidermal cell clusters that become segregated from the larval tissues.

These structures are called the imaginal discs, since they are going to give rise to most

of the adult insect body, also known as the imago (Fig.7). The imaginal discs follow a

developmental program distinct from that of their larval ‘host’, proliferating from 20‐

50 cells to a final size of 20000‐50000 cells 89. In particular, imaginal discs have been

preferred for the analysis of the combinatorial effects of many conserved signalling

molecules in the specification of positional values for differentiation and growth

(Fig.8). Because of its well characterized developmental circuitry and ease of dissection

I will focus on the wing imaginal disc as an epithelial model, and therefore I proceed to

briefly explain its major patterning events, as known to date.

The majority of the wing disc consists of a columnar epithelium, which will give

rise to most adult cuticular structures, being covered by a thin layer of squamous

epithelium called the peripodial membrane. The luminal face gives rise to the external

surface of the adult structures, following eversion of the disc during metamorphosis.

Specifically, the outmost region of the wing disc gives rise to the thorax structures (the

notum and the pleura), the next ‘ring’ will make the wing hinge, and the central pouch

of the disc will make the wing blade 89 (Fig.7).

Very early in larval development, an invaginating cell cluster that constitutes

the haltere and wing common precursor, inherits the apposed stripe pattern of

engrailed (en) and wingless (wg) expression from the anterior‐posterior (A‐P) boundary

of an embryonic parasegment.


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Fig.7 – Imaginal disc that will give rise to the adult appendages are present within the larvae. Fate

determination along the proximal to distal axis is believed to result from complex interactions between

the anterior‐posterior and dorso‐ventral morphogen‐mediated signalling, leading to the specification of

the wing blade, the wing hinge and the notum, that will give rise to the adult hemithorax.

In particular, the wing disc further derives from the dorsal part of the common

precursor, therefore inheriting a portion of the En stripe that specifies the posterior

compartment of the wing 90 (Fig.8). Although still elusive, distinct adhesive properties

between the posterior and anterior cells of the wing disc seem to prevent their

intermingling, therefore defining a clear compartment boundary, termed the A‐P

boundary. In the posterior compartment En seems to promote hedgehog (hg)

expression 91, 92, which acts as a short‐range morphogen to specify the expression of

decapentaplegic (dpp) in a narrow stripe of cells adjacent to the A‐P boundary 93

(Fig.8). Dpp constitutes a member of the TGF‐β family, promoting patterning and

differentiation of the wing beyond the central domain through a concentration‐

dependent mechanism, inducing the expression of target genes, such as spalt (sal),

optomotor‐blind (omb) and vestigial (vg) 94‐99. Besides giving genetic positional values

along the medial‐to‐lateral wing, the Dpp activity gradient seems to indirectly promote

wing growth 100, being counterbalanced by the brinker transcriptional repressor, which


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is expressed in a complementary lateral‐to‐medial fashion, being itself repressed by

Dpp activity 101, 102.

The important contribution of Dpp signaling to the proximal‐distal patterning of

the wing disc is further completed by intersection of signals coming from the dorsal‐

ventral (D‐V) boundary of the wing disc. During the early stages of larval development

the D‐V boundary is established by the antagonist relationship between dorsal

Epidermal Growth Factor Receptor (EGF‐R) signaling and ventral Wingless (Wg)

signaling, resulting in the expression of apterous (ap) in the dorsal region of the disc 90,

103 (Fig.8). Ap further specifies the activation of Notch at the D‐V boundary, through a

complex regulation of its ligands Serrate and Delta 104‐106. Notch is then assumed to

directly activate the transcription of genes at the D‐V boundary, including wg and cut,

which themselves further restrict the expression of the Notch ligands, fine‐tuning

Notch activity to a narrow stripe 107.

At the D‐V boundary Notch and Wg signaling synergistically specify the

development of the sensory cell precursors (SOPs) that will give rise to the margin

bristles. Furthermore, in the presumptive wing blade, the selector genes scalloped and

vestigial are transcriptionally activated by the combinatorial action of Notch, Wg and

Dpp signaling 108‐110 (Fig.8), providing the organizer functions of the D‐V boundary,

such as promoting growth along the proximal‐distal axis 111‐113, and specifying a

gradient of cell‐affinities that differentiates the presumptive blade from the hinge 114.

The combinatorial action of vestigial together with two of its target genes ‐ nubbin and

rotund ‐ seems to be required for the induction of a ring of Wg expression in the distal

hinge 115 (Fig.8). This is required for the proximal expression of the transcription factor

homothorax (hth), which together with teashirt (tea) is required for proper hinge

development 116, 117 (Fig.8). Furthermore, Homothorax provides an autoregulatory loop

that maintains the ring of wg expression essential for the normal growth of the hinge.

On the other hand, the iroquois gene complex (iro‐C), induced through dorsal EGF‐R

signaling and restricted to the notum‐hinge boundary by Dpp signalling 103, 118, 119

(Fig.8), is responsible for patterning the proximal and intermediary hinge structures,

providing cell‐affinity differences between the notum and hinge cells 120, 121. Most

proximally, pannier (pnr) and U‐shaped are critical for specifying notum dorsocentral

bristles through activation of wg expression in an anterior‐posterior stripe 122 (Fig.8).


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Fig.8 – Wing imaginal disc patterning. The domain of expression of some patterning molecules is

depicted from the most primordial stages of wing development (left) to the most advanced 3rd instar

imaginal disc (right).

In the end, this complex circuitry of signalling molecules seems to drive the

establishment and maintenance of a region specific cell architecture, possibly

providing cues for the modification of adhesive properties and cytoskeletal

architecture. This seems to occur mainly through the regulation of cell asymmetries

both in the apico‐basal axis and in the plane of the tissue, to which contribute the

amazingly complex array of adhesion molecules, polarity determinants and

cytoskeletal genes.


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II. Aims of the Project

The actin cytoskeleton is essential to support polarized vesicle transport within

epithelia and for endocytosis to take place. Most studies that have addressed these

issues used epithelial cell lines in culture. However, very few have analysed how actin

itself or specific ABPs are implicated to promote vesicle trafficking in vivo, in part,

because for most components of the actin cytoskeleton, their role within a tissue

remains obscure. In this project I aimed to: analyse whether the tumoral behaviour of

CP depleted cells results from Crb accumulation due to impaired endocytosis (Task 1);

analyse whether accumulation of other membrane surface receptors is evident in CP

mutant or depleted cells due to endocytic trafficking defects (Task 2); determine

whether loss of CP affect only a subset of vesicle trafficking events (Task 3) and finally

elucidate whether CP acts directly on vesicle trafficking or indirectly through

maintenance of a particular actin filament network (Task 4).


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III. Materials and Methods

1. Fly strains and genetics

1.1 Fly husbandry

All fly stocks were raised at 25oC, according to standard conditions 123. Crosses

were cultured in small vials containing a yeast‐glucose‐agar medium.

1.2 Fly Strains: mutant alleles and transgenic lines available for this study

The mutant alleles cpa107E and cpbM143 used during the course of this work are

likely null alleles, leading to a truncated protein lacking the actin‐binding domain,

since both have been shown to include stop codons in the beginning of the sequence

coding for the N‐terminal domain 124, 125. The majority of fly stocks used in this work

were available at the IGC, and some were kindly supplied by the labs of Ginés Morata

(CBMSO, Madrid) and António Jacinto (IMM, Lisbon). The Gal4 drivers and UAS‐

transgenes used in this study are reported in Table 1.

1.3 Fly Stocks generated

In order to robustly overexpress actin in wing imaginal discs, a recombinant line

was produced between the UAS‐act5C::GFP and UAS‐act42A::GFP 126, 127 transgenes,

both located on the second chromosome. Since the P‐elements used to insert both

transgenes carry the white (w+) marker, giving rise to an orange eye colour in a w‐

background, recombinant flies were selected for having a stronger, red eye colour. As

expected, driving the recombinant line with nub‐Gal4 gave rise to a enhanced wing

phenotype compared to flies overexpressing either UAS‐act5C::GFP or UAS‐

act42A::GFP alone (Sup. Fig.1).

To analyse Cpa colocalization with endosomes, the following transgenic lines w‐

; UAS‐Rab5::GFP/CyO, MKRS/ TM6β, w‐; UAS‐Rab7::GFP/CyO; MKRS/ TM6β were

crossed to w; sp/CyO; UAS‐HA::cpa/ TM2.

In order to evaluate a possible genetic interaction between CP and aPKC, two

independent P‐element insertions carrying the cpb RNAi constructs (w; UAS‐

cpbRNAiC10; MKRS/ TM6β and w; UAS‐cpbRNAiD4; MKRS/TM6β) were crossed with w;


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sp/CyO; UAS‐aPKCCAXXDN/TM6β, carrying a dominant negative version of aPKC to obtain

the line w; UAS‐cpbRNAiC10; UAS‐ DaPKCCAXXDN / TM6β. In addition, to induce clones of

cells mutant for either cpa or cpb that expressed the aPKCCAXXDN dominant negative

form, w; FRT42D, cpa107E/ CyO or w; FRT40A, cpbM143/ CyO flies were crossed to w; sp/

CyO; UAS‐aPKCCAXXDN/TM6 yielding w; FTR42D, cpa107E; UAS‐aPKCCAXXDN and w; FRT40A,

cpbM143; UAS‐aPKCCAXXDN, respectively.

Table 1 – Gal4 drivers and UAS constructs used during the course of this work.

Gal‐4 Drivers Spatio‐temporal domain of activity in the wing disc

engrailed‐Gal4 (en‐Gal4) 128

From end of embryogenesis to end of larval stage in the

posterior compartment (Fig.2)

nubbin‐Gal4 (nub‐Gal4) 129

From 2nd larval instar (48h) to end of larval stage in the

prospective blade and proximal hinge (Fig.2)

scalloped‐Gal4 (sd‐Gal4) 130

From 1st larval instar (24h) in the whole disc and restricted

in the blade and proximal hinge at 3rd instar larvae.

T155‐Gal4 131 From 1st larval instar (24h) in a patchy pattern

UAS‐transgene Purpose of use

UAS‐cpbRNAiC10 85

To promote knockdown of cpb expression

UAS‐act5C::GFP 127

To overexpress actin 5C tagged to GFP

UAS‐act42A::GFP 126

To overexpress actin 42A tagged to GFP

UAS‐crbstrong (minigen32.12C)42

To overexpress the entire crb coding region

UAS‐crbweak (minigen30.11d)42

To overexpress the entire crb coding region

UAS‐myc::crbintra 42

To overexpress the intracellular domain of crb

UAS‐rab5::GFP 133

As an early endosome GFP marker

UAS‐rab7::GFP 133

As a late endosome GFP marker

UAS‐HA‐cpa 84

To overexpress cpa tagged to an HA epitope

UAS‐aPKCCAXXDN 134

To express a ‘kinase‐dead’ dominant negative form of aPKC (DN) that is targeted to the membrane (CAAX)


20

1.4 Genetic tools

1.4.1 The UAS‐Gal4 system

Expression of UAS constructs was performed at 25oC, unless mentioned

otherwise, using the Gal4 system. The Gal4 protein is a yeast transcription factor that

activates GAL genes required for glucose metabolism. The Gal4 gene has been widely

cloned and used to genetically manipulate expression of genes in foreign organisms

such as Drosophila. Promoter sequences containing a consensus binding site for

activating Gal factors, termed UAS sequences, have been introduced in a number of

eukaryotic expression vectors, allowing for the production of transgenic constructs

that are conditionally expressed upon the availability of Gal4 135. Thus, in Drosophila, a

parental stock expressing Gal4 can be crossed to another carrying a UAS‐insert,

therefore driving expression of the later in the F1 progeny (Fig.1). Furthermore, several

well characterized enhancer sequences have been ‘trapped’ by the Gal4 coding gene

to yield spatio‐temporal specific drivers of gene expression 128.

1.4.2 Generation of clones by mitotic recombination

The FLP/FRT system was used to generate mitotic clones. The Flipase (FLP)

recombinase belongs to the family of λ integrases from Saccharomyces cerevisiae. The

members of this family are known to direct site‐specific recombination of two DNA

strands, which in the case of the FLP occurs through the recognition of Flipase

Recombination Target (FRT) sites. The FLP/FRT technique has been particularly

interesting for generating mosaics to study recessive mutations that would otherwise

cause embryonic lethality if present in the entire organism 136.

Several p‐element insertion stocks have been generated in Drosophila, introducing FRT

sites close to the centromere of each chromosome as well as a transgene encoding FLP

driven by a heat‐shock inducible promoter (hsp70). The fly stocks constaining FLP and

FRT sites, used during the course of this work to generate somatic clones, are indicated

in Table2. In this sense, mitotic recombination between FRT sites in homologues

chromatids occurs following a 1 hour treatment at 37oC of individuals at the stage of

interest. In this work, heat‐shock was performed at 1st and 2nd larval instar. As a result,

in a heterozygous setup, recombination between nonsiter chromatids results in the


21

generation of a daughter cell homozygous for the mutation distal to the FRT site.

Subsequent cell divisions of this homozygous mutant cell give rise to a mutant clone

that can be analyzed phenotypically. Conversely, from the same mitotic recombination

event, a wild‐type homozygous sibling cell is generated, giving rise to the twin spot of

the mutant clone. Usually, the wild‐type FRT chromatid contains a cell marker, such as

GFP under the control of a ubiquitous promoter. This allows for the generation of

negatively marked mutant clones (Fig.2).

Fig.1 – The Gal4 system in Drosophila. One of the parental stocks carries the Gal4 gene in close

proximity to a known enhancer of gene expression. This stock can be crossed to another containing a

UAS‐transgene, and the resulting progeny will express this transgene in the pattern of expression of the

Gal4 factor, which is specified by its associated enhancer region. A nubbin‐Gal4 line drives UAS‐GFP

expression in the presumptive wing pouch and distal hinge of the wing disc. The engrailed‐Gal4 line

drives UAS‐GFP expression in the posterior compartment of the wing disc.

en>UAScpbRNAiC10 nub>UAScpbRNAiC10

Phal Phal


22

On the other hand, mosaic analysis with a repressible cell marker (MARCM) was

used to positively mark mutant cpa and cpb clones, as well as to generate constitutive

expression of UAS‐transgenes in marked clones 137. The MARCM technique relies on

the combined use of Gal4 together with its repressor Gal80, arranged in a manner such

that FLP‐mediated mitotic recombination causes the genetic loss of Gal80 and the

concomitant derepression of Gal4. Such a derepression can be set to happen in a

mutant clone in a way that allows for the expression of a certain UAS‐marker, like UAS‐

GFP or another UAS‐transgene (Fig.2).

Table 2 – FLP/FRT system stocks used during the course of this work.

FLP/FRT stock Purpose of use

y‐,w‐,hsFLP122, UAS‐GFP; FRT42, tub‐Gal80;

tub‐Gal4/ TM6β 137

To generate cpa mutant clones, positively marked by GFP and expressing another UAS‐transgene.

y‐,w‐,hsFLP122, UAS‐GFP; FRT40, tub‐Gal80;

tub‐Gal4/ TM6β 137

To generate cpb mutant clones, positively marked by GFP and expressing another UAS‐transgene.

w‐; FRT42, tub‐GFP; T155‐Gal4, UAS‐FLP 138

To generate cpa mutant clones, negatively marked by GFP

2. Immunohistochemistry

Third instar larvae were dissected in 0,1M phosphate buffer (pH 7,2) and fixed in 4%

formaldehyde in PEM (0,1M PIPES pH 7,0; 2mM MgSO4; 1mM EGTA) for 30 minutes on

ice. As a standard protocol, except for anti‐Crb and anti‐Notch staining, larvae were

then washed and permeabilized in PBS 0,2% Triton X‐100 (PBT) for 15 minutes and

incubated with the primary antibodies diluted in PBT, supplemented with 10% donkey

serum, overnight at 4oC. The larvae were then washed 3 times, for 10 minutes each, in

PBT and incubated in the dark with secondary antibody diluted in PBT 10% donkey

serum, for 2 hours at 4oC.


23

Fig.2 ‐ Mitotic recombination induced by the FLP recombinase during the G2 stage of the cell cycle

results in the loss of heterozygosity at the locus coding for a mutation (black star) and a ubiquitously

expressed cell marker (green rectangle) (A). As a consequence one of the daughter cells will become the

precursor of the mutant clone, whereas the other will become the precursor of the complementary

‘wild‐type’ population (twin spot) that is marked by the presence of the cell marker, such as GFP (A). In a

MARCM set up, Gal80 represses the expression of a UAS‐insert (green rectangle), such as UAS‐GFP, but

loss of heterozygosity at the Gal80 containing locus, as induced by FLP mediated mitotic recombination,

results in expression of the UAS‐construct in the homozygous mutant clone (B).

GFP GFP

A B


24

For anti‐Crb and anti‐Notch staining, fixed larvae were previously incubated for

1 hour in a blocking solution of PBT 10% bovine serum albumin (BBT) and then

submitted to a quick dehydration‐rehydration series of 30%, 50% and 70% methanol in

BBT. This was found to greatly enhance the signal detection for membrane‐associated

proteins. The primary antibodies used were: mouse anti‐Arm, (N27A1, 1:10;

Developmental Studies Hybridoma Bank (DSHB)); rabbit anti‐activated Caspase3 (1:50;

Cell Signalling Techonology); mouse anti‐Crb (Cq4, 1:10; DSHB); mouse anti‐Notch

extracellular domain (C458.2H, 1:50; DSHB); goat anti‐Fat (1:1 cocktail of dR‐18 and

dC‐16, 1:20; Santa Cruz); rabbit anti‐Homothorax (1:500, a gift from Adi Salzberg);

mouse anti‐Cut (2B10, 1:5; DSHB); rabbit anti‐Rab5 (1:5000, a gift from Akira

Nakamura). The secondary antibodies used were donkey anti‐mouse and anti‐rabbit

conjugated to TRITC or Cy5, (1:200; Jackson Immunoresearch). Imaginal discs were

mounted in Glycerol 90%.

3. Phalloidin Staining

A phalloidin conjugated dye was used to stain F‐actin. Third instar larvae were

dissected and fixed as described in the previous section. Before mounting, tissues were

incubated with TRITC conjugated phalloidin, (1:200; Sigma), in PBT for 5‐8 minutes and

rinsed 3 times 10 minutes, before mounting in Glycerol 90%.

4. Image Acquisition and Analysis

Fluorescent images were obtained on a Zeiss LSM 510 Meta and Leica SP5

confocal microscope. Image files were processed using the Bitplane Imaris software®

to select standard z‐projections and optical cross‐sections through the epithelium. In

order to evaluate colocalization between fluorophores, binary colocalization masks

were obtained with the NIH Image J software®, using a cut off point of 50% relative to

maximum pixel intensity. Using this same software, confocal images destined for

colocalization analysis were previously submitted to standard medium filtering.


25

IV. Results 1. CP mutant and depleted cells accumulate the Notch receptor and the Crumbs polarity determinant in punctuated strucures

Lethal alleles of cpa were isolated in a previous mosaic genetic screen to

identify genes required for Drosophila eye differentiation 124. Given that capping of

actin filaments relies on a functional CP αβ‐heterodimer 21, 139, whose stability depends

upon the association of each subunit with one another 140, 141, loss of one subunit or

mutations preventing the association of both are assumed to effectively cause a loss of

capping activity. This is consistent with the accumulation of actin filaments observed in

either cpa or cpb mutant clones or in tissues depleted of cpa and cpb by RNA

interference, as well as with the fact that loss of either cpa or cpb gives rise to identical

phenotypes 84.

Interestingly, cpa107E mutant cells, undergoing extrusion in the wing blade

epithelium, mislocalize Arm and DE‐Cad to basolateral positions (data not shown),

whereas they accumulate the Crb polarity determinant in punctated structures

resembling vesicles (Fig.1). As the cpa and cpb extrusion phenotype had been shown

to be region specific, with mutant clones mainly extruding in the blade region of the

wing disc 84, I decided to evaluate whether Crb accumulation in cpa107E mutant clones

was also specific to the prospective blade, possibly accounting for the region specificity

of the cell extrusion phenotype. In order to observe cpa107E mutant clones within the

blade epithelium, I used a mosaic FLP/FRT system that uses an imaginal tissue‐specific

Gal4 driver (T155‐Gal4) to direct expression of the FLP enzyme, thus catalyzing a low

frequency of mitotic recombination during all stages of larval development 138.

Therefore, cpa107E mutant clones, marked by the absence of GFP, are produced in all

larval stages, which allows for the recovery of late induced mutant clones across the

whole wing disc of 3rd instar larvae. As expected, mutant clones induced in the

prospective blade region showed basal accumulations of Crb in the form of punctated

structures (Fig. 1). Interestingly, I observed similar accumulations in clones induced in

the presumptive hinge and notum (Fig.1), ruling out the possibility for a region specific

accumulation of Crb that correlated to the blade specific extrusion of cpa and cpb

mutant clones.


26

Fig.1 –cpa mutant clones induced in the wing imaginal disc accumulate Crb in the form of punctated

structures. cpa mutant clones are marked by the absence of nuclear GFP and Crb staining appears in red

(A); punctated Crb accumulations are seen throughout the tissue, matching GFP negatively marked

clones (A’‐AA’).

Distinct from what is observed in the clonal context, when broad regions of the

wing disc are depleted of cpb by RNAi, cells seem to undergo an epithelium to

mesenchymal‐like transition and overproliferate 85 . As expected, depletion of cpb by

RNAi using the eng‐Gal4 driver, leads to Crb accumulation. This not only occurs in

discrete punctuated structures resembling vesicles, but also at the level of apical peri‐

membrane clusters (Fig.2A‐A’’’). The same phenotype was observed, by depleting cpb,

using the sd‐Gal4 driver (Sup. Fig.2).

Accumulation of Crb at the cell membrane or in punctuated structures did not

resemble basolateral mislocalization of DE‐cadherin and Arm, that had previously been

observed in extruding cpa and cpb mutant clones 84. They were rather similar to

accumulations of membrane‐linked receptors observed in imaginal mutant tissue for

genes implicated in endocytic trafficking 67. Interestingly, similar to avl and rab5

mutant tissue, cpb depletion by RNAi, using en‐Gal4, also induces apical punctated

accumulation of the Notch extracellular domain, as indicated by anti‐Notchextra staining

(Fig.2B‐B’’’).

Crb GFP Crb

Crb GFP

A A’

A’’

cpa107E clones

cpa107E clones


27

Fig.2 – CP depleted cells accumulate the Notch receptor and Crb in the wing imaginal disc. cpb depleted cells are marked by the presence of GFP (green) and either Notch or

Crb appears in red/gray (A‐A’’’’, anti‐Notch; B‐B’’’’, anti‐Crb); Notch and Crb become accumulated in the posterior compartment as shown by anti‐Notch and anti‐Crb (gray),

respectively (A’’‐A’’’, B’’‐B’’’). Cross‐section views through the disc indicate that accumulation occurs at the level of intracellular punctated structures, and at the level of

apical peri‐membrane clusters (A’’’’‐ arrows; B’’’’ ‐ arrowhead). A – anterior, P – posterior

A P

A’’’’

A P

A’

A’’ A’’’

Crb

Crb GFP

Crb

Crb

Crb

A en>UAScpbRNAiC10

Notch

B B’

B’’ B’’’

B’’’’

Notch GFP

Notch GFP

Notch

Notch

en>UAScpbRNAiC10


28

Several membrane receptors and adhesion molecules, such as Notch, Fat,

EGFR, and E‐Cad have been shown to undergo endocytosis in wing disc cells through a

process that depends on the ERM proteins Merlin and Expanded 142. Therefore, in

order to determine whether other membrane associated proteins were accumulated

due to CP depletion, I analyzed the expression and subcellular localization of the

adhesion‐related proto‐cadherin Fat. Indeed, cpb depleted cells by RNAi accumulate

Fat, as indicated by anti‐Fat staining, although this seems to occur in a difuse manner

along the entire apical cell surface (Fig.3).

Fig.3 – CP depleted cells accumulate Fat in the wing imaginal disc. cpb depleted cells are marked by the

presence of GFP and Fat appears in red/gray (A‐A’’); Fat becomes apically accumulated in the posterior

compartment, as shown by anti‐Fat (gray) (A’‐A’’). A – anterior, P – posterior

Taken together, the data present here suggests that CP promotes endocytic

vesicle trafficking of Crb, Notch and Fat, or that it is able to restrict their transcriptional

upregulation. In order to test this hypothesis, I analyzed the expression levels of cut

and m8‐lacZ as a read‐out for Notch activity, since Notch upregulation induces

misexpression of these genes beyond the DV boundary 143, 144. Instead, expression of

cut and m8‐lacZ at the DV boundary, as indicated by anti‐Cut and anti‐β‐Gal staining,

A en>UAScpbRNAiC10

Fat GFP

A’

Fat

A P

A’’

Fat


29

respectively, is highly reduced in posterior wing cells depleted of cpb, indicating that

Notch signalling is disrupted (Fig.4). This suggests that Notch accumulation in CP

depleted cells does not result from overexpression of Notch, but that loss of CP

prevents Notch activation. It is therefore likely that accumulation of Notch, and

possibly of Crb and Fat, in cpb depleted cells, results from defects in endosomal

trafficking.

Fig. 4 – CP depleted cells show reduced expression of Notch target genes. cpb depleted cells are marked

by the presence of GFP (A) or by increased phalloidin staining (B); Cut and β‐Gal appear in blue/gray (A‐

A’‐ anti‐Cut; B‐B’ – anti‐β‐Gal); cut expression at the DV boundary is reduced in the posterior

compartment, as shown by anti‐Cut (gray) (A’); m8‐lacZ reporter expression at the DV boundary is

reduced in the posterior compartment, as shown by anti‐β‐Gal (gray) (B’).

Phall Cut GFP

Cut

A’

Phall β‐Gal (m8‐lacZ)

B

β‐Gal (m8‐lacZ)

B’

A en>UAScpbRNAiC10

en>UAScpbRNAiC10


30

2. Overexpression of the two cytoplasmic actin genes act5C and act42A can give rise

to hinge overgrowth and Crumbs accumulation.

Decreased expression of CP causes actin filament accumulation 84, 145. In this

sense, the putative role of CP in vesicle trafficking could be indirect, such that the actin

filament network formed in CP mutant cells would disrupt endocytic vesicle trafficking.

In order to understand whether actin filament accumulation, by itself, prevents

membrane and cytoplasmic accumulation of Notch and Crb, I’ve used the en‐Gal4 and

nub‐Gal4 drivers to overexpress the ubiquitous actin genes act5C and act42A, as their

overexpression promotes accumulation of actin filaments in the wing disc epithelium 146(Vinhas and Janody, unpublished). Interestingly, similarly to cpb depletion by RNAi,

overexpression of act5C, using en‐Gal4, leads to an expansion of the prospective distal

hinge, associated to accumulation of the apical polarity determinant Crb, as revealed

by anti‐Crb staining (Fig.5A). This accumulation occurs mainly in punctuated structures

at the apical cell surface (Sup. Fig.3). Furthermore, a relatively slight accumulation of

the Notch receptor also occurs in act5C overexpressing cells, as revealed by staining of

anti‐Notchextra (Fig.5B). Overall, this suggests that Crb and Notch accumulation,

following loss of CP, is caused by a global excess of F‐actin within the cell.

Surprisingly, overexpressing a recombinant version of UAS‐act5C::GFP and UAS‐

act42A::GFP, under the control of the nub‐Gal4 driver, comparatively enhanced the

hinge outgrowth observed in wing imaginal discs where each actin gene was

overexpressed individually (Fig.6). This suggests that hinge‐specific overgrowth

observed in CP depleted discs is attributable to actin filament accumulation, pointing

to a role of the cortical actin network in regulating growth processes in this region.

3. CP colocalizes with the early endosome marker Rab5

Punctuated accumulation of Notch, Crb and Fat in CP depleted tissue suggests

that CP might have a role in vesicle trafficking. In support of this, CP contains

hydrophobic segments that are likely to interact with membrane lipids 147. Therefore,

it is conceivable that CP could be in close contact with endocytic vesicles, which would

suggest a more direct role of CP in the regulation of endosome dynamics.


31

Fig.5 – Act5C overexpression leads to accumulation of Notch and Crb in the wing imaginal disc. cells

overexpressing act5C are marked by the presence of GFP and either Notch or Crb appear in red/gray (A‐

A’, anti‐Notch; B‐B’, anti‐Crb); Notch and Crb are accumulated at the apical cell membrane of posterior

depleted cells, as shown by anti‐Notch and anti‐Crb, respectively (A’, B’).

Fig. 6 – Actin overexpression induces hinge overgrowth in the wing imaginal disc. Actin overexpressing

cells are marked by the presence of GFP; phalloidin staining of F‐actin (red) allows for visualization of

cell architecture (A‐C); hinge outgrowths are observed in wing discs overexpressing either act42A or

act5C (A,B, ‐ arrows), although they look smaller than the ones observed in wing discs overexpressing

both act42A and act5C (C ‐ arrows). Note: expression of UAS‐constructs was performed at 22oC .

B

Phal Act5C‐GFP

C

Act42A‐GFP, Phal Act5C‐GFP

A

Phal Act42A‐GFP

nub>UASact42A::GFP nub>UASact5C::GFP nub>UASact5C::GFP UASact42A::GFP

B’

en>UASact5C::GFP A

Act5C‐GFP Notch

B en>UASact5C::GFP

Act5C‐GFP Crb

Notch

A’

Crb

B’


32

Interestingly, I found that an HA‐tagged form of cpa (HA‐cpa), which can fully

rescue the cpa and cpb mutant phenotype 84, apically localizes to punctated structures

resembling vesicles, as indicated by anti‐HA staining (Fig. 7A). Indeed, the punctated

pattern of early and late endosomes, labelled by Rab5‐GFP and Rab7‐GFP, respectively,

strongly resembled in size and position the pattern outlined by staining of HA‐Cpa

(Fig.7A‐C). Taking this into account, I’ve qualitatively analysed colocalization between

early or late endosomes marked by the presence of Rab5‐GFP and Rab7‐GFP,

respectively, and HA‐Cpa. Colocalization masks, above 50% of pixel intensity, revealed

a punctated pattern of colocalization between Rab5‐GFP marked early endosomes and

HA‐Cpa (Fig.7D‐D’’). However, a similar pattern of colocalization was not verified

between Rab7‐GFP marked late endosomes and HA‐Cpa (Fig.7E‐E’’). Furthermore,

optical cross sections through the wing imaginal discs expressing rab5::GFP or

rab7::GFP, and HA::cpa, also suggest colocalization of early endosomes with the apical

punctated pattern of HA‐Cpa (Fig.7D’’’), whereas late endosomes appear to be

localized more basally (Fig.7E’’’).

This suggests that CP directly associates with early endosomes, possibly being

relevant to the regulation of early endosome dynamics. Indeed, in cpb depleted cells

by RNAi, using the en‐Gal4 driver, I’ve observed an increased abundance of Rab5‐

positive puncta, as indicated by anti‐Rab5 staining (Fig.8). This further suggests that

early endosomes become accumulated in CP depleted cells, being either unable to

recycle back to the plasma membrane or to traffic to the lysosome.

4. crb overexpression in the wing imaginal disc gives rise to different cellular

outcomes depending on the tissue genetic context

Vesicular accumulation of Crb has been coupled to neoplastic tumor

development of avl mutant tissue 67. Interestingly, CP depletion by RNAi in the distal

wing hinge induces hyperplastic tissue overgrowth, while in the wing blade epithelium,

most cells seem to delaminate and possibly overproliferate (Janody, unpublished).

Moreover, similar to cpa and cpb mutant clones, avl mutant clones are extruded from

the wing blade epithelium 67.


33

Fig.7 – Cpa partially colocalizes with early endosomes. Clones overexpressing HA‐cpa (A), rab5::GFP (B)

and rab7::GFP (C) reveal apical vesicular structures (green) that are similar in size and shape (Arm (red)

outlines the apical cell membrane) (A‐C). Early and late endosomes (green) are marked by the presence

of Rab5‐GFP (D) and Rab7‐GFP (E), respectively; anti‐HA (red) reveals a punctated pattern of HA‐Cpa

localization (D’, E’); colocalization masks above 50% of pixel intensity (white) reveal partial colocalization

of HA‐Cpa (red) with Rab5‐GFP (green) (D’’), but not with Rab7‐GFP (green) (E’’); confocal cross sections

trough the wing imaginal disc show that HA‐Cpa partially colocalizes with Rab5‐GFP marked early

endosomes (green) (D’’’), but not with Rab7‐GFP marked late endosomes (green) (E’’’).

A

B

C

Arm Rab5‐GFP

Arm HA‐Cpa

Arm Rab7‐GFP

D nub>UAScpbRNAiC10; UAS‐rab5::GFP

D’

D’’

D’’’

HA

Rab5‐GFP

E nub>UAScpbRNAiC10; UAS‐rab7::GFP

E’

E’’

E’’’

HA

Rab7‐GFP


34

Fig.8 ‐ CP depleted cells accumulate Rab5‐positive endosomes in the wing imaginal disc. cpb depleted

cells are marked by the presence of GFP and Rab5 appears in red/gray (A‐A’’). Rab5‐positive puncta

apically accumulate in the posterior compartment, as shown by anti‐Rab5 (gray) (A’‐A’’). A – anterior, P

– posterior

In order to test whether an excessive Crb signalling activity could justify

extrusion of cpa and cpb mutant clones, I’ve induced clones overexpressing crb in the

wing imaginal disc, assuming that if Crb accumulation promotes extrusion of CP

mutant cells, groups of cells overexpressing crb should behave in a similar manner. To

induce clonal overexpression of crb, I’ve made use of the MARCM system. Accordingly,

in a clonal context, crbintra overexpression leads to cell death, as revealed by the

expression of activated Caspase 3 in GFP‐positive cells (Fig.9A‐A’’,B‐B’’). However,

dying cells are mostly observed in the presumptive wing blade epithelium, defined by

the folding formed between the presumptive blade and hinge regions and revealed

with anti‐Arm staining, which marks the apical cell membrane (Fig. 9A,A’’,B,B’’).

Optical cross‐sections through the wing disc showed that crb overexpressing cells

extrude basally in presumptive blade, whereas some cells can often be recovered in

the hinge and notum regions (Fig. 9B’’’). This clonal behaviour is reminiscent to the cpa

and cpb extrusion phenotype, suggesting that extrusion of CP mutant cells could be

linked to Crb accumulation.

A’’

Rab5 GFP Rab5

A en>UAScpbRNAiC10 A’

A P


35

Fig.9 – crb overexpressing clones extrude mainly in the presumptive wing blade. crbintra overexpressing

clones are marked by the presence of GFP (green) (A‐B’’’); caspase3 positive cells (blue) are present

mainly in the wing blade (A’’, B’’), although some can also be observed in the wing hinge (B’’‐ arrow);

many clones are still maintained in the wing hinge (B’’, B’’’ – arrows). The dashed line is limiting the

boundary between the presumptive wing blade and hinge. b – blade, h ‐ hinge

To confirm that homotypical Crb overexpression can lead to a tumoral

phenotype, I overexpressed crb using the nub‐Gal4 driver. Under these conditions, crb

overexpression does indeed induce the formation of epithelial cysts and numerours

foldings (Fig.10). Larvae overexpressing crb (crbweak) develop wing discs that look

mostly hyperplastic, where the epithelial sheet looks overexpanded, particularly in the

presumptive hinge (Fig. 10A). On the other hand, overexpression of a similar but

stronger contruct (crbstrong), leads to the development of neoplastic wing discs that are

mainly comprised of round cells (Fig.10B).

A A’ A’’

B B’ B’’

B’’’

Arm GFP C3

Arm GFP C3

Arm GFP C3

C3

C3

b h

b h

GFP

GFP

UAScrb intra clones

UAScrb intra clones


36

Fig.10 – crb overexpression in a broad tissue context leads to tumoral development of the wing imaginal

disc. Cell architecture is visualized with phalloidin staining of F‐actin in red/gray (A‐D). Epithelial

transformation in the range of neoplastic (A,C) to hyperplastic (B) is observed in crb overexpressing

discs, as compared to wild‐type (D); crbweak overexpressing discs, reveal an expansion of the distal hinge

(B‐ arrow); crbintra overexpression gives rise to neoplastic phenotypes, revealing epithelial cysts that form

preferentially in the distal hinge (C‐ arrow); confocal cross section reveals mutlilayering of the tissue (C’)

as compared to wild‐type (D’).

Furthermore, larvae overexpressing the intracellular domain of crb (crbintra)

show severe neoplastic phenotypes, leading to the formation of epithelial cysts

(Fig.10C). Optical cross sections through the wing disc epithelium show that the tissue

architecture is dramatically altered, producing an intricate plane of folding (Fig.10C’).

Phal

D WT

Phal

C nub>UAS‐crbintra

D’PhalPhal

C’

A nub>UAS‐crbweak

Phal Phal

B nub>UAS‐crbstrong


37

5. crb overexpression in the wing imaginal disc induces hinge specific overgrowth

Surprisingly, I realised that the prospective wing blade, in proximity to the DV

compartment boundary, is less affected by crbintra overexpression driven with nub‐

Gal4, when compared to the surrounding outsized hinge tissue (Fig.10C; Fig.11). Cells

neighbouring the DV boundary have a normal shape, maintaining the proper tissue

organization, while the presumptive hinge cells assume a round shape, which is

concomitant with a variable loss of cell polarity manifest in folding of the tissue

(Fig.10C‐C’; Fig.11A’). This phenotype was confirmed using the rn‐Gal4 driver to

overexpress crbintra (data not shown). Thus, similar to CP loss, the crb overexpression

outcome seems to depend on tissue identity. In order to confirm this, I stained crb

overexpressing wing discs driven by nub‐Gal4, for Homothorax (Hh), whose normal

pattern of expression is restricted to the hinge and notum regions, and for Wg, which

is normally expressed in a narrow stripe of cells along the DV boundary and in two

concentric rings in the wing hinge. I did find that Hth is expressed in severely

unpolarized tissue (Fig.11B’’’), indicating that these cells have derived from a hinge‐

defined cell lineage or that they have acquired hinge genetic fate. Expansion of Wg

expression domain was also detected in the prospective hinge of crb overexpressing

discs, but not at the DV boundary (Fig.11A’’,B’’). Curiously, overgrowth observed in CP

depleted tissues also seemed to arise mainly from the hinge region (introduction

Fig.6E), suggesting that this fate defined epithelial region is more susceptible to loss of

proliferation control.

Cell death is usually suppressed in many tumoral processes, although a small pool

of dying cells is always reported, possibly as footprint of cell competition 73. Wing discs

overexpressing cbrintra show only a few cells stained positively for the activated form

of Caspase3, meaning that the cell death program is only mildly activated throughout

the tissue, at least during this stage of development (Fig.11A’’’). The same was verified

for cpb depleted wing discs by RNAi, where the majority of the overgrown tissue does

not express the activated form of Caspase3 148.


38

Fig. 11 – crbintra overexpression induces overgrowth of the presumptive wing hinge. Cell architecture is visualized with phalloidin staining of F‐actin (red) (A‐A’, B‐B’);

Caspase3‐positive cells (blue) are few and sparse, arising mainly between severely unpolarized tissue (A, A’’’); Wg domain of expression (green) is broadened and

disorganized in the presumptive hinge (A,A’’,B, B’’), although it looks unaffected at the DV boundary (A’’) or absent if the presumptive wing blade has suffered a great

reduction (B’’), as compared to wild‐type (C); Hth domain of expression (blue) is broadened towards the central prospective blade (B,B’’’), as compared to wild‐type (D).

A’’’

C3

C WT

Wg

Hth

D WT

A

Phal Wg C3

B

Phal Wg Hth

A’

Phal

B’’

Wg

B’’’

Hth

B’

Phal

A’’

Wg

nub>UAS‐crbintra

nub>UAS‐crbweak


39

6. CP genetically interacts with aPKC to define cell shape and prevent cell extrusion

A ‘kinase‐dead’ dominant negative form of a‐PKC (aPKCCAXXDN) has been shown

to reduce the activity of overexpressed crb and could partially suppress the polarity

and proliferation phenotypes of imaginal discs depleted of avl 67. Actually, when I

drove crbintra and aPKCCAXXDN in the wing imaginal disc, using the nub‐Gal4 driver, I

could rescue pupal lethality associated to crbintra overexpression (Sup. Fig.4). If the CP

phenotype is a consequence of Crb accumulation, I expected that expressing

aPKCCAXXDN in CP depleted cells should prevent cells from losing polarity and

overproliferate. Surprisingly, clones mutant for cpa107E or cpbM143 and expressing

aPKCCAXXDN, using the MARCM system, not only undergo extrusion and apoptosis in the

prospective wing blade, but also in the hinge and notum epithelia, as revealed by

staining of activated Caspase 3 in GFP positive cells. (Fig.12B). A cross section through

the wing blade epithelium showed that GFP‐positive cells undergo extrusion in all

regions of the wing disc (Fig.12B’). On the contrary, clones of cells expressing

aPKCCAXXDN alone are perfectly maintained within the epithelium and show no evidence

of polarity disruption, as indicated by normal apical localization of Arm (Fig.12A‐A’’).

Since expression of aPKCCAXXDN has a dominant negative effect, this suggests that CP is

absolutely required to maintain aPKC deficient cells within the epithelium tissue.

Consistent with an enhancement of the CP depletion phenotype, driving both

aPKCCAXXDN and cpb depletion using nub‐Gal4, results in a severely enhanced adult wing

phenotype, when compared to the phenotype of wings depleted of cpb alone (Fig.

13D, D’, D’’). The former show partial or total loss of vein differentiation, hair

misorientation, disorganization of the margin sensory bristles and an overall necrotic

and blistered appearance (Fig.13C, C’, C’’), while expression of aPKCCAXXDN alone has no

visible effect on wing morphogenesis (Fig.13B, B’, B’’). Moreover, cpb depletion by

RNAi along with expression of aPKCCAXXDN, using the en‐Gal4 driver, seems to induce

cell roundening and progressive loss of the columnar epithelial shape, which is evident

by the fact that cells in the posterior wing compartment look shorter along their apical‐

basal axis than anterior cells serving as an internal control (Fig.14C’,D’). This is further

associated to a significant reduction of the posterior wing compartment, suggesting a

reduction in cell size (Fig.14C,D). Cell size reduction is also evident in cpb depleted


40

wings expressing aPKCCAXXDN, using nub‐Gal4, since wing hairs are much reduced when

compared to the hairs of wings depleted of cpb alone (Fig.12C’’, D’’).

All together, these results suggest that CP and aPKC work in parallel to prevent

epithelial cell extrusion and to promote and maintain the columnar cell architecture

typical of wing disc epithelial cells.

Fig. 12 – CP genetically interacts with aPKC to prevent loss of epithelial cell polarity and extrusion.

Clones expressing aPKCCAXXDN are marked by the presence of GFP (green) (A‐A’); cpbM143 mutant clones

expressing DN‐aPKC are marked by the presence of GFP (green) (B‐B’); anti‐Arm staining (red/gray)

outlines the apical cell membrane (A‐B’’); clones expressing aPKCCAXXDN don’t show any polarity or

adhesion defects, as indicated by anti‐Arm staining (A‐A’’); cpb mutant cells expressing aPKCCAXXDN

extrude in all regions of the wing disc epithelium, including the wing hinge and notum (B‐B’‐ arrows),

and many undergo apoptosis, as indicated by anti‐activated Caspase 3 staining (blue) (B‐B’).

Arm GFP C3 B’

B’’

Arm

Arm GFP C3

B

Arm GFP C3 A’

Arm GFP C3

A

A’’

Arm

UAS aPKCCAXXDN clones

UAS aPKCCAXXDN , cpbM143clones


41

Fig. 13 – CP genetically interacts with aPKC during wing morphogenesis. cpb depleted wings (C) are

smaller than wild‐type (A); expressing aPKCCAXXDN (B‐B’’) has no effect on wing morphogenesis, as

compared to wild‐type (C‐C’’); wings depleted of cpb and expressing aPKCCAXXDN are reduced and have

loss of vein differentiation (D), as compared to wings depleted of cpb alone (C); cpb depleted wings

show an increased density and disorganization of wing margin bristles (C’), which becomes enhanced by

expressing aPKCCAXXDN at the same time (D’), as compared to wild‐type (C’); wing hairs look misoriented

in cpb depleted wings (C’’), becoming densely distributed in cpb depleted wings expressing aPKCCAXXDN

(D’’), as compared to wild‐type (A’’).

B’’

A’ B’ C’ D’

A’’

C’’ D’’

A WT C nub>UAS‐RNAicpbC10 B nub>UAS‐aPKCCAAXDN D nub>UAS‐RNAicpbC10; UAS‐aPKCCAAXDN


42

Fig.14 – CP genetically interacts with aPKC to maintain the epithelial columnar cell shape. No visible

enhancement of cpb depletion phenotype (A) was observed by expressing aPKCCAXXDN (B), in the nubbin

domain of expression; the posterior wing compartment is much reduced (D) and cells seem to become

more cuboidal than their anterior counterparts (D’), when aPKCCAXXDN is expressed in cpb depleted

tissue, as compared to wings depleted of cpb alone (C‐C’).

nub>UAS‐cpbRNAiC10 nub>UAS‐cpbRNAiC10 UAS‐aPKCCAAXDN

PhalPhal

Phal

A’

B’

A B

Phal


43

VI. Discussion

1. The role of CP in endocytosis

Endocytosis and polarized exocytosis seems to be crucial for the establishment

and maintenance of epithelial cell polarity. I found that loss of CP in the Drosophila

wing imaginal disc leads to accumulation of the membrane associated proteins, Crb

Notch, and Fat (results Fig.1‐3). In particular, Notch accumulation is unlikely to be due

to transcriptional upregulation, since Notch strongly accumulates at the cell surface of

CP depleted cells but remains inactive, as suggested by downregulation of Notch target

genes (results Fig.4). Alternatively, accumulation of Crb and Notch is suggestive of

endocytic trafficking defects, since apical Notch and Crb levels have been shown to be

regulated by endocytic lysosomal degradation 67, 149.

In fact, my results suggest that CP is likely to regulate early endosome

dynamics. Foremost, I found that an HA‐tagged form of Cpa co‐localized with the early

endosomal marker Rab5‐GFP, but not with the late endosomal marker Rab7‐GFP

(results Fig.7). Additionally, I found that Notch signalling is downregulated following

loss of CP. Interestingly, Notch is endocytosed upon ligand binding and is transferred

to late endosomes where activation can proceed through cleavage by the λ‐Secretase

complex 149‐151. Accordingly, Notch accumulation in late endosomes leads to its

ectopic activation, such as in vps25 mutant tissue 63, 64, 152. However, when lysosomal

targeting is disrupted, Notch accumulates at the cell surface but is unable to signal,

such as in avl and Rab5 mutant tissue 68. In this sense, it is likely that Notch

accumulation in CP depleted cells is due to an early endocytic fusion defect, blocking

progression to late endosomes were activation of Notch takes place. Accordingly,

accumulation of Crb and Fat in CP depleted cells could also be due to an endocytic

defect.

Growing evidence indicates that the actin cytoskeleton plays a fundamental

role in endocytic trafficking 59, 60, 153. In this sense, the role of CP in early endosome

dynamics could be mediated through its well‐known function in promoting actin

filament termination. Indeed, I found that actin filament accumulation could lead to

defects in vesicle trafficking, since actin overexpression (act5C and act42A) leads to


44

Notch and Crb accumulation, as was observed in CP depleted cells (results Fig.5, Sup.

Fig.3). As a fact, the apical actin web in epithelial cells seems to be crutial for the

transport of vesicles, since cytocalasin treatment, which depolymerised actin filaments

in this region, inhibits endocytosis 54. Furthermore, the actin filament cross‐linking

protein Spectrin, which localizes to the apical actin web, was shown to promote

endocytosis in enterocytes of the Drosophila gut epithelium 154. In this sense, actin

filament accumulation, due to CP depletion or actin overexpression, could lead to

filament bundling and disruption of the cross‐linked network of the apical actin web,

therefore preventing endocytosis.

In detail, strong evidence suggests that actin polymerization at sites of vesicle

assembly occurs by recruitment and activation of the Arp2/3 complex 155‐158.

Furthermore, the adaptor protein CD2AP, which regulates Arp2/3 activity at the level

of early endosomes, is able to bind CPs, in vitro 159. Thus, the combined activity of

Arp2/3 and CPs could produce a specific actin structure that is required to assist in the

early steps of vesicle formation (Fig.1A‐B). Additionally, a structural scaffold, such as

the apical actin web, might be needed to stabilize the local membrane composition of

RabGTPase effectors, arranging them in a precise architectural layout to allow for

directed vesicle fusion 160. Therefore, apart from regulating early steps of vesicle

formation, CPs could generate an actin framework required to stabilize oligomeric

protein complexes that promote directed docking and fusion events towards the

degradative or recycling pathways (Fig.1C).

To summarize, I propose that short actin filaments, terminated by the activity

of CPs, are required for the initial steps of vesicle formation and possibly for providing

a structural support for directed vesicle fusion events during endocytosis (Fig.1).

2. Endocytosis defects might contribute to growth of CP mutant tissue

Interestingly, CP depleted cells seem to overproliferate, and this could be

related to defects in internalization and lysosomal degradation of membrane

receptors. In fact, blocking the endocytic pathway induces overproliferation and can

lead to the formation of neoplastic tumors 62, 66. Curiously, neoplastic development

and overproliferation of avl or rab5 mutant cells was shown to be coupled to Crb


45

vesicular accumulation, leading to loss of cell polarity as well to overproliferation 67.

Indeed, in a homotypical environment, crb overexpressing cells, as well as CP depleted

cells, are able to survive or even overproliferate in the distal hinge region (results

Fig.10). Therefore, Crb accumulation could at least be partially responsible for the

overgrowth of CP depleted tissue.

Fig.1 – Actin filament networks generated by the barbed end capping activity of CP could contribute to

the assembly and pinch‐off of endocytic vesicles (A‐B), as well as to oligomerize protein complexes in

vesicle membrane domains (C), to specify docking towards endocytic recycling or degradation.

In addition, my results revealed that CP depleted cells also accumulate the Fat

tumour suppressor gene (results Fig.3). There are at least two signalling branches

downstream of Fat, one concerning the establishment of planar cell polarity (PCP) and

requiring the transcriptional corepressor Atrophin 161, and another restricting cell

proliferation through the Hippo pathway 162‐164. The fact that CP depletion gives rise to

hinge overproliferation and to misorientation of the wing hairs indicates that Fat

signalling is blocked at an early stage, before giving rise to the PCP and Hippo signalling

branches (Fig.2).

A

B

C


46

The conserved Hippo signalling pathway has been proposed to regulate contact

inhibition of growth 165, 166, being mediated by the kinase complexes Hippo/Salvador

and Warts/Mats and culminating in the phosphorylation and consequent inactivation

of the transcriptional co‐factor Yorkie (Yki) 163, 167‐169. By inactivating Yki, the Hippo

signalling cascade prevents transcription of growth promoting genes, such as cyclin E

and wg, as well as anti‐apoptotic genes, such as diap1 170, 171. Furthermore, it prevents

transcription of upstream components of the pathway, such as expanded (ex), in a

negative‐feedback loop 172 Indeed, disruption of Hippo signalling seems to occur in CP

mutant or depleted cells, since these upregulate ex and wg (Janody and García‐

Fernandez, unpublished).

Interestingly, mutant clones for components of the Hippo signalling cascade

show defects in endosomal trafficking, and this has been correlated to disruption of

the Notch signalling pathway 173, 174. Furthermore, Merlin (Mer) and Expanded (Ex),

two upstream members of the Hippo pathway, have been shown to colocalize with

vesicle compartments and regulate endocytosis of numerous transmembrane proteins,

including Notch and Fat 142. However, whether they do so independently of Yki

transcriptional regulation is still a matter of debate 142, 175. My results show an

extensive colocalization of CP with early endocytic compartments (results Fig.7),

suggesting that CP has a direct role in regulating vesicle trafficking rather than an

indirect role through its effect on Hippo signalling. In this sense, CPs could cooperate

with Ex and Mer to regulate endocytosis of Notch and Fat, independently of Hippo

signalling (Fig.2). However, it still needs to be addressed whether CP plays a role in

Hippo signalling through its putative influence on endocytosis, or if it does so

independently of vesicle trafficking (Fig.2).

Despite the reported similarities, CP mutant or depleted cells show additional

phenotypes that differ from those of mutants for components of the Hippo pathway.

For instance, mer and ex mutant cells shown apical accumulation of E‐cadherin 142, 175,

whereas CP mutant cells essentially mislocalize it to basolateral positions. Additionally,

the hyperplastic overgrowth of tissue mutant for components of the Hippo pathway

does not match the polarity disruptive overgrowth of CP depleted tissue. Thus, besides

potentially promoting Hippo signalling, CPs could independently regulate the stability

of cell‐cell adhesion and polarity.


47

Fig.2 – CP and the ERM proteins Mer and Ex, could cooperate to promote steady‐state clearance of

membrane receptors. This could eventually contribute to the regulation of signalling pathways, such as

the Fat/Hippo signalling pathway.

3. crb overexpression leads to cell competition in a heterotypical tissue environment

Upon CP depletion, the accumulation of Crb occurs everywhere along the wing

disc epithelium. Although crb overexpression might be far from mimicking punctated

Crb accumulation in CP mutant cells, clones overexpressing this polarity determinant

extrude mainly in the presumptive wing blade, as do CP mutant clones (results Fig.9),

suggests that cells do not respond identically to Crb levels along the proximal‐distal

axis. On the other hand, crb overexpression in a broad domain of the wing epithelium

leads to tissue overgrowth (results Fig.10). This is in agreement with the definition of a

process of cell competition, according to which, interaction between ‘loser’ and

‘winner’ cells leads to elimination of the loser population, whereas ‘loser’ cells

wouldn’t die if present in a homotypical environment 73.

Endocytic internalization of survival factors could be one of the principles by

which cell competition is implemented on a population basis 176. For instance, clones

mutant for endocytic components such as Rab5, avl and vps25 seem to be eliminated

through a process of cell competition, despite the abnormal overgrowth they produce


48

in a homotypical environment 61, 67, 152. Given that CP seems to be involved in endocytic

trafficking of membrane receptors, abnormal endocytic mechanisms could lead to

defective internalization of a survival factor in CP mutant cells, causing cell extrusion

and death in the clonal context (Fig.3A). It has been suggested that, in the wing

imaginal disc, the limiting ligand for endocytosis is the TGF‐β homologue Dpp 177.

However, a defect in Dpp uptake is not sufficient to explain the region preferential

extrusion of CP mutant cells, since overexpression of a constitutive active form of tkv

in CP mutant clones does not rescue the extrusion phenotype (Janody, unpublished).

Nevertheless, this does not rule out the possibility that endocytosis defective cells

might not respond identically along the proximal‐distal axis of the wing epithelium. In

fact, Rab5 mutant clones seem to be preferentially eliminated in the presumptive wing

blade 68,178. Therefore, it is possible that internalization of other factors, besides Dpp, is

critical to prevent cell extrusion in the presumptive blade.

Another possible mechanism by which cells could initiate cell competition is by

exchanging information through cell‐cell communication mechanisms 179. A

mathematical model predicts that build‐up of negative pressure around a group of

cells leads to their distortion and may trigger apoptosis in the clonal context 180. In this

sense, stronger adhesive interactions in the distal wing epithelium could constrain

cells, preventing the flow that relieves local pressure and thereby accounting for the

specificity of the clone extrusion phenotype in this region (Fig.3B). In fact, apical cell

shapes and expression of DE‐cad are graded in response to Wg secreted from the DV

organizer, pointing to stronger adhesive interactions in the distal wing epithelium 181.

Interestingly, CP mutant clones induced in the hinge and notum are rarely

extruded but develop smooth borders, evidencing affinity differences in relation to

their WT neighbours (Supp.Fig.5). These apparently conflicting processes could

represent a continuum of responses that depends on the strength of the affinity

differences along the proximal‐distal axis of the wing disc. For instance, crb

overexpressing clones in the hinge and notum seem to round up before extruding,

possibly minimizing their adhesive contacts with WT neighbours.

Overall, my results suggest that CPs and crb could cooperate to prevent cell

extrusion, and that disruption of actin dynamics or polarity maintenance respectively

mediated by these two proteins can lead to cell competition.


49

Fig.3 ‐ Cell extrusion of CP mutant clones and crb overexpressing cells could be due to defective

endocytic uptake of survival factor (A). Alternativly, a mechanical stress imprinted by the disequilibrium

of adhesive tension forces between these cells and there WT neighbours, could trigger cell extrusion

and apoptosis (B).

4. The Crb complex and the actin cytoskeleton might cooperate to prevent growth

distortions in the wing imaginal disc

Consistent with the existence of a proximal‐distal gradient of cell affinities, crb

overexpression and CP depletion seems to induce hinge specific overgrowth, which

could be due to reduced resistance of this tissue to cell contact inhibition, such as

mediated by the Hippo pathway.

To activate Hippo signalling, CPs could link actin dynamics at the cell periphery

to cortical tension sensing between adhesive neighbours, thereby transducing a

growth contact‐inhibitory signal. For instance, CP mutant and depleted cells, as well as

crb overexpressing cells, look rounder then WT, possibly pointing to a role in regulating

A B


50

cortical stiffness. Interestingly, increased membrane stiffness seems to be required for

isometric cortex contraction and microtubule spindle attachment during mitosis 182, 183.

Therefore, increased cell roundness in CP depleted and crb overexpressing cells could

disturb the maintenance of uniform disc growth. Indeed, upon CP depletion or actin

overexpression, wing imaginal discs seem to grow disproportionally, showing size

enhancement of the peripheral regions belonging to the distal hinge (introduction

Fig.6E; results Fig.10). A mathematical model explains that growth in the centre of the

disc could be induced by the combined activity of Dpp and a second growth factor

coming from the D‐V boundary, whereas growth in the peripheral regions would be

induced by stretching due to central pouch growth 184. In this sense, autonomous and

non‐autonomous growth mechanisms seem to operate along the wing epithelia, the

non‐autonomous being highly dependent on cell‐cell interactions, possibly through

adhesion molecules, such as Fat 164.

Surprisingly, I’ve shown that crb overexpression in a broad tissue context drives

hinge specific neoplastic transformation. This situation is associated with expression of

the proximal fate determinant Hth and with disturbance of the Wg morphogen ring in

the distal hinge (results Fig.11). Upregulation of Wg in this region could be due to

defective Fat signaling activity, since Wg is upregulated in Fat mutant clones in the

distal hinge 185, and disruption of Fat activity leads to the transcriptional upregulation

of glypincans 186, which are able to increase the extracellular diffusion of Wg 187, 188.

Furthermore, crb overexpression seems to induce a dramatic expansion of the

presumptive hinge (results Fig.11), supporting the idea that the Wg morphogen

promotes growth in this region 189. Alternatively, distal cells overexpressing crb could

dedifferentiate and reacquire the ability to express Hth, therefore contributing to

hinge proliferation. Indeed, Hth can be looked as a primordial transcription factor,

being expressed in proliferating and undifferentiated imaginal discs and being

ultimately repressed by distal specific transcription factors, such as Vg in the wing disc 116, 117.

Interestingly, my results show that neoplastic transformation of the

presumptive blade upon crb overexpression seems to be prevented or delayed near

the D‐V boundary, since loss of the normal epithelial structure is less apparent in the

cells apposed to it (results Fig.11). This could mean that some factor emanating from


51

the D‐V organizer prevents neoplastic transformation by reinforcing polarity and

adhesion. Interestingly, Wg limits cell proliferation in the presumptive wing blade,

despite its growth promoting activity in the wing hinge 189. Therefore I hypothesize

that, despite the potential deregulation of Fat signalling in CP depleted and crb

overexpressing cells, the growth inhibitory function of Wg at the level of the D‐V

boundary is unaffected, leading to resistance of the presumptive blade region to

overproliferation.

5. The actin cytoskeleton might be crucial to reinforce the maintenance of epithelial

surface integrity

Despite the striking differences in the resulting cell morphology, similar growth

patterns of CP depleted and crb overexpressing tissue, suggested that CP and crb could

genetically interact to regulate tissue growth. Instead, by combining CP depletion with

expression of a kinase‐dead aPKC to reduce Crb polarizing activity, the adult wing

phenotype was severely enhanced. Although this does not rule out a possible genetic

interaction between crb and CP, it revealed a previously unreported genetic

interaction between CP and aPKC in the regulation cell size and shape.

The role of aPKC during the establishment and maintenance of cell polarity is

likely complicated. For instance, embryos maternally and zygotically mutant for

aPKCk06403, affecting the kinase domain, show apical clustering and punctated

accumulation of DE‐cad and Par‐3, which is concomitant with embryonic epithelial

polarity defects at the onset of gastrulation 190. This was attributed to abnormal

cytoskeleton interactions that arise from unattachment of microtubules from the

centrosome during cellularization 190. However, another aPKC mutant, also affecting

the kinase domain, has been isolated that shows no evidence of a zygotic phenotype

given the maternal contribution of a ‘WT’ aPKC allele, although it recapitulates the

gastrulation‐defective phenotype if ‘WT’ contribution is not provided by the female

germline (Ferreira, Prudêncio and Martinho, unpublished). This seems to indicate that

some function of aPKC is required for the establishment of epithelial polarity and

adhesion during early embryonic development, but is not longer crucial for

maintaining it during later stages. Accordingly, my results show that clonal expression


52

of a kinase‐dead aPKC during the 2nd and 3rd larval instar doesn’t seem to affect the

polarity and adhesion of wing imaginal disc cells.

Interestingly, expression of a kinase‐dead aPKC in cpa (data not shown) or cpb

mutant clones leads to cell extrusion and death in all regions of the wing imaginal disc

(results Fig.12), suggesting that polarity maintenance still relies on aPKC during larval

stages. In fact, it has been suggested that the actin cytoskeleton plays a crucial role in

providing tension at junction remodelling sites, such as during cell rearrangements that

establish the ommatidial cores in the Drosophila retina 191. As such, CPs could be

crucial to promote the formation of an actin network that provides tension and

prevents cell extrusion whenever AJ stability is compromised, such as in the case of

aPKC loss of function.

Wings depleted of CP and expressing aPKCCAAXDN show an increased density of

wing hairs and margin bristles as well as shortening of the cells’ apical‐basal length,

while expression aPKCCAAXDN alone gives no obvious phenotype in the adult wing

(results Fig.13‐14). This suggests a synergistic role of CPs and aPKC in regulating cell

size and shape. At least in some cases, the apical Baz/Par‐3/PAR‐6/aPKC complex is

thought to locally regulate cytoskeletal dynamics, through the Rho‐like GTPase Cdc42

and Rac. Furthermore, Rac seems to suppress Rho activity, thereby preventing

excessive acto‐myosin contractility 192. In the Drosophila wing epithelium, Rac1 is

essential for actin polymerization at the level of the AJ, and Cdc42 is important for cell

elongation and reorganization of the basal actin network 193. In detail, localized apical

activity of Cdc42 and Rac could promote the activity of the Arp2/3 complex 194, 195,

thereby promoting actin filament polymerization and cell elongation. On the contrary,

Rho1 has recently been shown to promote isometric cortex contraction thereby

promoting transition from the columnar to cuboidal cell shape (Widmann and

Dahmann, unpublished). In cells depleted of CP, the aPKC complex might be sufficient

to promote the columnar shape, through activation of the Arp2/3 complex (Fig.4).

However, if CP is lost in cells expressing a domainat negative aPKC, the actin network

typical of anisotropic columnar cells might be disrupted, favouring the cuboidal shape

(Fig.4). In these sense, CP activity in concert with the Arp2/3 complex would be

sufficient to produce the actin network typical of columnar epithelial cells, preventing

transitions to the cuboidal shape that would lead to tissue shape distortions (Fig.4).


53

Fig.4 – Synergy between aPKC and CP in the definition of epithelial cell shape. aPKC regulates actin

dynamics through the Rho‐like GTPases Cdc42 and Rac, which, in turn, activate the Arp2/3 co‐factors,

SCAR/WAVE and WASP, respectively. Rho activity might antagonize the Arp2/3 complex and CPs in the

production of anisotropic cortical tension, promoting isometric cortex contraction of the acto‐myosin

network and transition to the cuboidal cell shape (A). Upon expression of a dominant negative aPKC

(DN‐aPKC), Arp2/3 modulation by Cdc42 and Rac might become interrupted, which could lead to a

columnar‐to‐cuboidal cell shape transition, unless CP activity is able to compensate for the generation of

anisotropic tension (B, C).

A B

C


54

7. Concluding remarks

Overall, the data presented in this thesis has contributed to understand the

role of CPs in endocytic vesicle trafficking of membrane surface receptors.

Furthermore, this work has contributed to clarify a role of the actin cytoskeleton and

the Crb polarity complex in the control of uniform and coordinated tissue growth

during development, the disruption of which could result in tumorigenesis. This work

has pointed to a significant synergy between between the actin cytoskeleton and

polarity/adhesion complexes in achieving structural support for cell shape changes and

adhesion during epithelial morphogenesis.

In the near future, understanding the signal transducing capacity of the actin

cytoskeleton, from the regulation of vesicle trafficking to mechanotransdution of

cortical tension, will help us understand how this cellular constituent impinges on

growth, and possibly differentiation pathways. In particular, combining new

biophysical approaches with genetic and biochemical data will help us understand how

cells sense the physical aspects of their environment, thereby triggering signaling

pathways through force‐bearing actin filaments.


55

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Appendix I


64

Drosophila melanogaster as a model organism in developmental biology – a brief

historical background

Drosophila melanogaster, commonly known as the ‘fruit fly’, has become one

of the most widely used animal models in biological research. Dating from the first

decade of the twentieth century, Thomas Hunt Morgan’s laboratory set the basic

techniques for breeding and identifying large collections of mutants, allowing for the

development of a new era in genetics and developmental biology 196.

Due to its short life cycle, spanning only 12 days at normal room temperature,

and to the ease and low cost of its culturing techniques, Drosophila became a favourite

model in the utmost years of genetic research. The field of Drosophila genetics had its

major development taking advantage of the use of balancer chromosomes, phenotypic

markers and non‐occurrence of meiotic recombination in the male sex. For instance,

balancer chromosomes were artificially generated by multiple inversions and

translocations of the wild‐type chromosomes, allowing for the inhibition of

homologous recombination in mutant and transgenic stocks, so that these can be

maintained without selection. On the other hand, phenotypic markers, either

dominant or recessive, allow tracking of their associated chromosomes over multi‐

generation crosses, so that certain genotypes of interest can become easily selected

and scored.

Since the first large scale generation of mutants, many phenotypic aspects,

such as the denticle pattern of the embryonic cuticle, turned out to be an excellent

read‐out to support systematic screens, pertaining to identify new genes and their

epistatic processes 197. However, it was not until the 1980s that recombinant DNA

technology developed in bacteria and phage opened new ways to target genes and

regulatory sequences and to further observe patterns of expression. The ingenious

domestication of wild transposable elements has allowed the use of vector mediated

transformation by transposon sequences, such as P‐elements in Drosophila, and

became the major technique in the generation of animal transgenics through many

forms of genome engineering 198, 199.

Even more, mosaic tissue analysis of particular mutations, through the artificial

induction of mitotic recombination, has allowed for the study of complex interactions


65

between genetically heterogeneous tissues, giving new insights on the specificity and

instructive nature of phenotypes, which are not easily evaluated in the drastic context

of whole mutant animals. More recently, powerful genetic tools, such as the use of the

UAS‐Gal4 and MARCM systems, allowed for the conditional expression of genes in

Drosophila, giving us a better understanding of gene function in time and space132.

Since the publication of its euchromatic genome in 2000, Drosophila became

known as an important reference for human biological research, for instance sharing

over 70% of the proteins involved in human disease 200‐202. Additionally, in this new

century, Drosophila research continues to expanded its use of technological advances,

such as knockdown RNA interference, microarrays, confocal imaging, laser ablation

and many others, therefore being a major subject in biological research and leading to

the discovery of evolutionarily conserved developmental mechanisms in other

metazoans.


66

Appendix II


67

Suplementary Figures Sup.Fig.1 – Overexpressing both act42A and act5C in the wing imaginal disc leads to additional wing

defects, as compared to the phenotypes of wings overexpressing act5C or act42A alone (C‐D).

Ovexpressing either act5C or act42A leads to notching of the wing margin (B‐C), although this is slightly

more evident in the case of act5C overexpression (C), as compared to WT (A); simultaneous

overexpression of both act5C and act42A leads to more severe defects, including: the appearance of

necrotic patches (D – red arrowhead), loss of hinge structures and the differentiation of ectopic blade

tissue, disrupting the proximal‐anterior margin (D – arrow).

Sup. Fig.2 – CP depleted cells accumulate the Notch receptor and Crb in the wing imaginal disc. cpb

depleted cells are marked by the presence of GFP (green) and Crb appears in red/gray (A‐A’’’); Crb

accumulates at the apical cell surface (A’’) and at the level of punctated structures (A’‐A’’’ – arrow).

A WT B nub>UAS‐act42A C nub>UAS‐act5C D nub>UAS‐act42A, UAS‐act5C

sd>UAS‐cpbRNAiC10

A A’

A’’

A’’’

Crb GFP Crb

Crb

Crb


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Sup. Fig.3 – Overexpression of act42A and act5C leads to accumulation of Crb in the wing imaginal disc.

Cells overexpressing act42A (A), act5C (B), or both act42A and act5C (C,D) are marked by the presence

of GFP (green), and Crb appears in red/gray (A‐D’’); Crb accumulates at the apical cell surface in wing

discs overexressing either act42A (A’), act5C (B’) or both act42A and act5C (C’), as indicated by anti‐Crb

(gray); Crb also accumulates at the level of puncated structures, as indicated by anti‐Crb (gray), in wing

discs overexpressing both act42A and act5C (D’‐D’’).

A’

Crb

B’

Crb

C’

Crb

D D’ D’’

Crb Crb Crb GFP

nub>UASact42A::GFP A

Crb At42A‐GFP

nub>UASact5C::GFP B

Crb Act5C‐GFP

nub>UASact5C::GFP UASact42A::GFP C

Crb Act42A‐GFP Act5C‐GFP


69

Sup. Fig.4 – aPKC positively regulates the intracellular domains of Crb, during wing morphogenesis.

Driving crbintra and DN‐apkc in the wing imaginal disc gives rise to adult wings that show some mild

defects: loss of cross‐vein differentiation and general broadening of the blade surface (B), as compared

to WT (A).

Sup. Fig.5 – cpa107E and cpbM143 mutant clones induced in the wing imaginal disc have reduced contact

with their tissue surroundings, suggesting differences in cell‐cell affinity towards their WT neighbouring

cells. Measurement of clonal Ferret’s circularity is based on the estimation of a single distance, the

Ferret’s diameter, which corresponds to the largest diameter of an irregular object (the clone). This

formula gives the ratio between the object’s area (A) and the area of a disc reference with the same

Ferret’s diameter (π(dFer./2)2 (varies between 0>Fer. Circ. >1, such that values close to 0 approach the

perfect square circularity, and values close to 1 approach the perfect disc circularity). cpa107E and cpbM143

are significantly rounder than ‘WT’ clones induced in mutation‐clear FRT42 and FRT40 isogenic lines,

respectively (p‐value cpa107E = 0,0028, N = 32; p‐value cpbM143 = 0,0099, N=36). Clones of each

genotype are shown in B‐E, being marked by the absence of GFP (green). NOTE: clones were induced at

1st and 2nd larval instars. A – area, dFer – Ferret’s diameter.

D FRT42D clones

GFP

B FRT42D, cpa107E clones

GFP

A WT B nub>UAS‐crbintra; UAS‐aPKCCAAXDN

A C FRT40A, cpbM143 clones

GFP

E FRT40A clones

GFP


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Experimental genotypes

Results Fig.1 ‐ GFP‐ cpa‐ clones: w‐; FRT42D, tub‐GFP/FRT42D, cpa107E; T155‐Gal4, UAS‐

FLP .

Results Fig.2‐4, 8 ‐ express UAS‐cpbRNAiC10 in the en domain: w‐; en‐Gal4, dll‐lacZ/

UAS‐cpbRNAiC10; UAS‐mCD8::GFP .

Results Fig.5 ‐ express UAS‐act5C::GFP in the nub expression domain : w‐; nb‐Gal4/

UAS‐act5C::GFP .

Results Fig.6 ‐ express UAS‐act5C::GFP in the nub expression domain : w‐; nb‐Gal4/

UAS‐act5C::GFP (A); express UAS‐act42A::GFP in the nub expression domain: w‐; nb‐

Gal4/ UAS‐act42A::GFP (B); express UAS‐act5C::GFP, UAS‐act42A::GFP in the nub

expression domain: w‐; nb‐Gal4/ UAS‐act42A::GFP, UAS‐act5C::GFP (C).

Results Fig.7 ‐ clones expressing HA‐cpa: y‐,w‐,hsFLP122; FRT42D, tub‐Gal80/FRT42D,

UAS‐HA‐cpa (A); clones expressing rab5::GFP: y‐,w‐,hsFLP122; FRT42D, tub‐

Gal80/FRT42D, UAS‐rab5::GFP (B); (C) clones expressing rab7::GFP: y‐,w‐,hsFLP122;

FRT42D, tub‐Gal80/FRT42D, UAS‐rab7::GFP; (D‐D’’’) express UAS‐rab5::GFP and UAS‐

HAcpa in the nub expression domain: w‐; nb‐Gal4/ UAS‐rab5::GFP; UAS‐HAcpa; (E‐E’’’)

express UAS‐rab7::GFP and UAS‐HAcpa in the nub expression domain: w‐; nb‐Gal4/

UAS‐rab7::GFP; UAS‐HAcpa.

Results Fig.9 – clones expressing UAS‐crbintra: y‐,w‐,hsFLP122, UAS‐GFP; FRT42D, tub‐

Gal80/ FTR42D, UAS‐crbintra; tub‐Gal4.

Results Fig.10‐11 ‐ express UAS‐crb in the nub expression domain: w‐; nb‐Gal4/ UAS‐

crbweak (A); w‐; nb‐Gal4/ UAS‐crbstrong (B); w‐; nb‐Gal4/ UAS‐crbintra (C‐C’).

Results Fig.12 ‐ (A‐A’) clones expressing UAS‐aPKCCAAXDN: y‐,w‐,hsFLP122, UAS‐GFP;

FRT40D, tub‐Gal80/ FRT40A; tub‐Gal4/ UAS‐aPKCCAAXDN; (B‐B’) cpb‐ clones expressing

UAS‐aPKCCAAXDN: y‐,w‐,hsFLP122, UAS‐GFP; FRT40D, tub‐Gal80/ FRT40A, cpbM143; tub‐

Gal4/ UAS‐aPKCCAAXDN.

Results Fig.13 ‐ (B‐B’’) express UAS‐aPKCCAAXDN in the nub expression domain: w‐; nub‐

Gal4/sp; UAS‐aPKCCAAXDN; (C‐C’’) express UAS‐cpbRNAiC10 in the nub expression

domain: w‐; nub‐Gal4/ UAS‐cpbRNAiC10; (D‐D’’) express UAS‐cpbRNAiC10 and UAS‐

aPKCCAAXDN in the nub expression domain: w‐; nub‐Gal4/ UAS‐cpbRNAiC10; UAS‐

aPKCCAAXDN.


71

Results Fig.14 ‐ (A) express UAS‐cpbRNAiC10 in the en expression domain: w‐; nb‐Gal4/

UAS‐cpbRNAiC10 ; (B) express UAS‐cpbRNAiC10 and UAS‐aPKCCAAXDN in the en

expression domain: w‐; nb‐Gal4/ UAS‐cpbRNAiC10; UAS‐aPKCCAAXDN.

Sup. Fig. 1 ‐ express UAS‐act42A in the nub expression domain: w‐; nub‐Gal4/UAS‐

act42A (B); express UAS‐act5C in the nub expression domain: w‐; nub‐Gal4/UAS‐act5C

(C); express UAS‐act42A and act5C in the nub expression domain: w‐; nub‐Gal4/UAS‐

act42A, UAS‐act5C (D).

Sup. Fig.2 ‐ express UAS‐cpbRNAiC10 in the sd expression domain: sd‐Gal4; UAS‐

cpbRNAiC10; UAS‐mCD8::GFP

Sup. Fig.3 –express UAS‐act42A in the nub expression domain: w‐; nub‐Gal4/UAS‐

act42A (A‐A’); express UAS‐act5C in the nub expression domain: w‐; nub‐Gal4/UAS‐

act5C (B‐B’); express UAS‐act42A and act5C in the nub expression domain: w‐; nub‐

Gal4/UAS‐act42A, UAS‐act5C (C‐D’’).

Sup. Fig.4 ‐ express UAS‐crbintra and UAS‐aPKCCAAXDN in the nub expression domain: nub‐

Gal4; UAS‐crbintra; UAS‐aPKCCAAXDN.

Sup. Fig.5 ‐ GFP‐ cpa‐ clones: y‐,w‐,hsFLP122; FRT42D, tub‐GFP/FRT42D, cpa107E (B) ; GFP‐

cpb‐ clones: y‐,w‐,hsFLP122; FRT40A, tub‐GFP/FRT40A, cpbM143 (C); GFP‐ WT clones y‐,w‐

,hsFLP122; FRT42D, tub‐GFP/FRT42D or y‐,w‐,hsFLP122; FRT40A, tub‐GFP/FRT40A (D‐E).

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