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A targeted gain-of-function screen identifies genes affecting salivary gland

morphogenesis/tubulogenesis in Drosophila

Vanessa Maybeck*1 and Katja Röper*

*Department of Physiology, Development and Neuroscience, University of

Cambridge, Cambridge CB2 3DY, UK

1current address: Institute of Neuroscience and Biophysics, Molecular Biophysics

(INB-2), Research Center Jülich, D-52425 Jülich, Germany

Genetics: Published Articles Ahead of Print, published on December 8, 2008 as 10.1534/genetics.108.094052

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Running title: Salivary gland morphogenesis in Drosophila

Keywords: morphogenesis, salivary glands, tubulogenesis, cell shape, cytoskeleton

Author for correspondence: Katja Röper,

Department of Physiology, Development and

Neuroscience,

University of Cambridge,

Downing Street,

Cambridge CB2 3DY, UK

Phone: ++44 (0)1223 333542

Fax: ++44 (0)1223 333840

email: kr250@cam.ac.uk

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ABSTRACT

During development individual cells in tissues undergo complex cell shape changes

to drive the morphogenetic movements required to form tissues. Cell shape is

determined by the cytoskeleton and cell shape changes critically depend on a tight

spatial and temporal control of cytoskeletal behaviour. We have used the formation

of the salivary glands in the Drosophila embryo, a process of tubulogenesis, as an

assay to identify factors that impinge on cell shape and the cytoskeleton. To this end

we have performed a gain-of-function screen in the salivary glands, using a

collection of fly lines carrying EP-element insertions that allow the overexpression of

downstream-located genes using the UAS-Gal4 system. We used a salivary gland

specific fork head-Gal4 line to restrict expression to the salivary glands, in

combination with reporters of cell shape and the cytoskeleton.

We identified a number of genes known to affect salivary gland formation,

confirming the effectiveness of the screen. In addition we found many genes not

implicated previously in this process, some having known functions in other tissues.

We report the initial characterization of a subset of genes, including chickadee,

rhomboid1, egalitarian, bitesize, and capricious, through comparison of gain and

loss-of-function phenotypes.

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INTRODUCTION

During development and organogenesis most tissues arise from layers of epithelial

cells that reorganize through complex morphogenetic movements. Many adult

organs consist of tubular arrangements of epithelial sheets, and these tubules form

during development through a process called tubulogenesis. There are a number of

ways to generate tubules (LUBARSKY and KRASNOW 2003). One important process is

the direct conversion of epithelial sheets into tubules through wrapping (COLAS and

SCHOENWOLF 2001) or budding (HOGAN and KOLODZIEJ 2002). Cells undergoing

tubulogenesis change their shapes drastically, from a cuboidal or columnar

epithelial shape to a wedge-shape or conical, and then back to a more columnar

epithelial shape once positioned inside the tube. Cell shape is determined by the

intracellular cytoskeleton, primarily actin and microtubules. The cytoskeleton is

closely coupled to cell-cell adhesion as well as adhesion to the extracellular matrix.

We are interested in understanding how the cytoskeleton and thus cell shape is

regulated and coordinated during tubulogenesis.

We chose to perform a gain-of-function screen rather than a mutagenesis-

based loss-of-function screen as phenotypes observed in the latter might be subtle

and thus missed or phenotypes in a given tissue might be obscured by disruption of

other tissues and many genes might also have redundant functions. In contrast the

gain-of-function/overexpression approach allows a particular tissue and gene to be

targeted, and many such screens have been successfully conducted in the past (for

examples see: BEJARANO et al. 2008; MOLNAR et al. 2006; RØRTH et al. 1998). The

screen presented here uses the formation of the salivary glands in the Drosophila

embryo as an assay system. The screen is based on a collection of transposable

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elements (EP elements) generated by Rørth (RØRTH et al. 1998) that contain UAS

sites that respond to the yeast transcription factor Gal4 followed by a promoter

directing expression, when activated, of genes located downstream 3’ of the EP

insertion site. If combined through crosses with a tissue specific source of Gal4

(HENDERSON and ANDREW 2000; ZHOU et al. 2001) overexpression (and in some

cases antisense expression) of a downstream gene will be activated only in the

target tissue, in our case the embryonic salivary glands in the Drosophila embryo.

Salivary gland formation in Drosophila is probably the simplest form of

tubulogenesis (LUBARSKY and KRASNOW 2003). A patch of about a two hundred cells

in the ventral epidermis of the embryo within parasegment two becomes specified to

form a salivary gland primordium, the placode, with a hundred cells on either side of

the embryo. This fate determination occurs through a combination of the activities of

the homeotic genes sex combs reduced (scr), extradenticle (exd), homothorax (hth),

and dorsal signalling by decapentaplegic (dpp) (HENDERSON and ANDREW 2000;

HENDERSON et al. 1999; PANZER et al. 1992). Without scr, exd, hth function, no

salivary glands form. Different subpopulations of cells are found in the invaginated

gland, such as the secretory cells, and the common and individual duct cells. Their

distinction depends on EGF signalling from the ventral midline (HABERMAN et al.

2003; KUO et al. 1996). Once the cells have been specified at stage 10 of

embryogenesis no further cell division occurs within the primordium, and no cells

are lost through apoptosis (BATE and MARTINEZ ARIAS 1993; CAMPOS-ORTEGA and

HARTENSTEIN 1985; MYAT and ANDREW 2000a). Invagination initiates in the dorsal

posterior corner of the primordium, with all future secretory cells invaginating in a

precise order, followed by invagination of the duct cells and formation of the ducts

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(MYAT and ANDREW 2000b). A key gene essential for the invagination is fork head

(fkh). Fkh is a winged-helix transcription factor, and in its absence all of the cells

fated to form the glands remain on the surface of the embryo as they fail to undergo

apical constriction (MYAT and ANDREW 2000a). Once inside the embryo, the glands

have to navigate their way through the surrounding tissues including the visceral

mesoderm and CNS to reach their extended final position parallel to the midline and

anterio-posterior axis. They are guided by cues from the surrounding tissues

(HARRIS and BECKENDORF 2007; HARRIS et al. 2007; KOLESNIKOV and BECKENDORF

2005). Also, after initially invaginating in a posterior-dorsal direction, the glands turn

and further extend into the embryo in a direction parallel to the anterio-posterior

embryonic axis, in a process dependent on integrins and downstream signals

(BRADLEY et al. 2003; VINING et al. 2005).

A few factors have previously been identified that impinge on the

cytoskeleton and cell shape during salivary gland morphogenesis. The actin

cytoskeleton is modified through proteins such as Btk29/Tec29 in conjunction with

Chickadee (CHANDRASEKARAN and BECKENDORF 2005). Small GTPases such as Rac

and Rho affect the invagination of the glands (PIRRAGLIA et al. 2006; XU et al. 2008).

Crumbs and Klarsicht affect the delivery of apical membrane and thus cell shape at

late stages of morphogenesis (MYAT and ANDREW 2002). Nonetheless, how these

factors work together throughout the whole process of invagination is still not clear

and it is likely that many others remain to be identified.

We have performed a gain-of-function/overexpression screen in the salivary

glands with the aim to firstly identify more genes that are required for salivary gland

tubulogenesis (and thus potentially also for tubulogenesis in general) and to

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secondly use this system as an assay for factors affecting cytoskeleton and thus cell

shape in general. The first aim assumes that genes that have a function in the

morphogenesis of the glands and are endogenously expressed in the glands might

perturb their invagination if overexpressed and if levels of expression are important.

The second aim hypothesizes that overexpression of genes not endogenously

expressed in the glands but important for cell shape coordination in other tissues will

lead to identifiable phenotypes in this screen, as defects in cell shape changes

resulting from the overexpression will affect the proper invagination of the glands.

The orientation of some of the EP elements is also likely to lead to (over)expression

of an antisense RNA, thus potentially inducing a tissue-specific loss-of-function

effect. We identified seven genes that have previously been implicated in salivary

gland morphogenesis or function confirming the effectiveness of the screen, and

also 44 insertions that uncover genes with potentially novel roles in the salivary

glands or functions in the regulation of cell shape and the cytoskeleton in other

tissues. Of these genes 14 are previously uncharacterized genes. A selection of the

genes that fall into the three categories discussed above (i.e. overexpression of a

gene with a function in the glands, overexpression of a gene not expressed in the

glands, revealing a function in cell shape coordination in other tissues, and loss-of-

function of a gene through tissue-specific antisense RNA expression) recovered in

the screen is examined in more detail below, including bitesize, egalitarian,

chickadee, capricious and rhomboid1.

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RESULTS

Experimental design of the gain-of-function screen

In order to address how the cytoskeleton and cell shape is regulated during such a

process of tubulogenesis, we performed a gain-of-function screen in the salivary

glands of the Drosophila embryo. We used a gland-specific Gal4-driver, fkhGal4

(HENDERSON and ANDREW 2000; ZHOU et al. 2001), to drive expression of either a

marker of the microtubule cytoskeleton, GFP-EFGas2 (SUBRAMANIAN et al. 2003), or

a marker of cell shape, SrcGFP (KALTSCHMIDT et al. 2000) in the glands only. Flies

carrying these marker chromosomes (GFP-EFGas2 or SrcGFP maker plus fkhGal4;

‘marker line’) were crossed to a collection of EP-element lines containing UAS-

elements, leading to the expression of gene X located 3’ downstream of the EP-

element insertion site. We drove expression from 1001 EP elements specifically in

the salivary glands and screened for any apparent problems in their morphogenesis

(see Fig. 1 for wild-type morphogenesis and marker expression, and Fig.2A for a

scheme explaining the screen set-up). It has previously been shown that the proper

invagination and positioning of the salivary glands depends on the surrounding

tissues such as the visceral mesoderm (VINING et al. 2005). The tissue-specific

expression of genes in the screen allowed us to identify factors that acted within the

glands themselves and did not affect functioning of the surrounding tissues, thus

giving a phenotype due to a secondary defect.

We crossed flies of ‘marker to flies carrying an EP-insertion on the second or

third chromosome (see scheme in Fig. 2A). The resulting offspring overexpressed a

gene X specifically in the salivary glands. These embryos were collected ‘early’

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(stage 10-13) and ‘late’ (stage 13-15) during embryogenesis and analyzed live for

any apparent defect in salivary gland invagination, positioning, and shape of salivary

gland cells or gland lumen. When phenotypes were observed in more than 20% of

embryos, embryos were collected again, fixed and counterstained for actin using

phalloidin to analyze general morphology. Positive insertions were defined as

having 20-90% of embryos showing a salivary gland phenotype after the second

examination. The baseline rate of obtaining salivary glands with a phenotype in

embryos expressing GFP-EFGas2 or SrcGFP in the glands under the control of

fkhGal4 was ~4% (see Material and Methods). In some cases the position and

orientation of EP-elements would be predicted to lead to the overexpression of an

antisense RNA rather than coding sense mRNA. Positive insertions resulting from

presumed antisense RNA expression are indicated as such in Table 1 below.

Phenotypes identified in the screen

We screened 1001 EP element insertions on the second and third chromosome (a

list of all lines screened can be found in the Supplementary Table 1).

Overexpression in the salivary glands of genes located downstream of EP elements

under the control of fkhGal4 led to a variety of phenotypes that could be classified

into four major classes (Fig. 2): invagination defects (‘failure to invaginate’, ‘wide

invagination’; see Fig. 2 B, C), gland shape and lumen defects (‘shepherd’s crook’,

‘C-shape’, ‘S-shape’, ‘wrong length’, ‘enlarged lumen’, ‘aberrant lumen’; see Fig. 2

D-I), positioning defects (‘wrong position’, ‘turning’, ‘budding’, ‘forking’, ‘hook’,

‘butterfly’; see Fig. 2 K-P) and gland sub-fate defects (‘no proper duct’; see Fig. 2Q).

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These overall phenotypes suggest that the screen detected interference at all

stages of salivary gland formation.

Although the phenotypes listed above were recurrently found in the screen,

half of the positive insertions showed a variable phenotype, combining several of

these individual phenotypes. The other half showed a consistent phenotype

restricted to one class or even one specific phenotype (see Table 1, ‘Phenotype in

Salivary Glands’). This suggests that in the cases of genes showing variable

phenotypes upon overexpression in the glands, a specific phenotypes was not

necessarily reflective of only a certain process failing during the invagination, but

rather indicates that many perturbances at the molecular level might lead to similar

phenotypes.

Genes identified in the screen

Out of 1001 EP lines screened 51 showed a phenotype in the salivary glands when

crossed to fkhGal4, equalling 5.09% of the total lines analyzed. The penetrance of

phenotypes varied from weak (2.7% of EPs tested), to strong (1.7% of EPs tested)

and very strong (0.7% of EPs tested; see Table). The genes affected could be

classified according to their predicted function (see Table 1). Several of the genes

identified by positive insertions have previously been implicated in salivary gland

morphogenesis or function within the glands: chickadee (CHANDRASEKARAN and

BECKENDORF 2005), tec29 (CHANDRASEKARAN and BECKENDORF 2005), doughnut on

2 (HARRIS and BECKENDORF 2007), rhomboid1 and spitz (KUO et al. 1996), tapδ

(ABRAMS and ANDREW 2005), and slit (KOLESNIKOV and BECKENDORF 2005). Three of

the insertions identifying these were potentially inducing antisense RNA expression

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and could thus mimic a loss-of-function situation (chic, tec29, slit), three insertions

would induce overexpression (dnt, rhomboid1, spitz). Overexpression of dnt could

affect the positioning cues the migrating glands receive, whereas rhomboid1 and

spitz overexpression would lead to excess Spitz ligand being provided, potentially

overstimulating EGFR signalling (see below). These genes served as confirmation

that factors impinging on salivary gland morphogenesis were picked up in this

screen.

The majority of positive insertions (44 out of 51 EPs), though, were inserted

into genes that have not previously been implicated in salivary gland morphogenesis

or tubulogenesis in general. Several of the encoded proteins have known functions

in other tissues in flies such as Egalitarian (NAVARRO et al. 2004), Traf-4 (CHA et al.

2003), RanGAP (KUSANO et al. 2001), Smd3 (SCHENKEL et al. 2002), Nedd 8 (ZHU et

al. 2005) and Tout-velou (THE et al. 1999). Fourteen out of the 51 hits were EPs

inserted in previously uncharacterized genes, many with close orthologues in other

species including vertebrates.

Analysis of individual genes in detail

In the following section we will discuss a subset of the genes identified in the

screen. Three of these were genes endogenously expressed in the glands

(chickadee, rhomboid1, and egl), and thus overexpression could have interfered

with their proper function in the glands. One gene (btsz) was expressed in the

glands and was identified through an insertion that would have lead to production of

a tissue-specific antisense RNA, thus potentially mimicking a loss-of-function

situation. The last gene (caps) was not endogenously expressed in the glands and

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thus the overexpression identified a potential requirement elsewhere for proper cell

and tissue shape.

Genes endogenously expressed in glands identified through overexpression:

Chickadee encodes the Drosophila Profilin protein. Profilins are actin-

monomer sequestering proteins, that have been implicated in promoting both actin

polymerization or depolymerization (YARMOLA and BUBB 2006). Drosophila Profilin

fulfils essential functions at all stages of development, and also in the female

germline (VERHEYEN and COOLEY 1994). Loss of profilin has been associated with

the inability to constrict apical surfaces in the morphogenetic furrow of the larval eye

disc (BENLALI et al. 2000). With respect to salivary gland morphogenesis it has been

reported that tec29 chic double mutants show disorganized actin in the salivary

gland placode and display a delay in invagination (measured by remaining placode

area at stage 14). This study also reports that chic mutant embryos have normal

glands (CHANDRASEKARAN and BECKENDORF 2005). Two EP insertions into chic

showed phenotypes in the glands when driven by fkhGal4, EP(2)713 and

EP(2)1011. EP(2)713 should overexpress the entire chickadee coding sequence

(see supplementary Figure 1), whereas EP(2)1011 is inserted in the opposite

direction and could drive expression of an antisense RNA to the 5’ most 1kb of the

chic pre-mRNA (or it could drive expression of eIF4a, situated 1.8kb away, see

scheme in Fig. 3A). Expression of either EP led to aberrantly shaped glands (Fig.3

B-E), with EP(2)1011 giving frequent hook-shaped and ‘shepherd’s crook’-shaped

glands (Fig.3 D and E). Overexpression of a UAS-chickadee construct using

fkhGal4 led to embryos showing a disorganized epidermis in the regions where

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fkhGal4 was expressed, with a loss of apical Crumbs accumulation in the epithelial

cells of the epidermis (Fig. 3 F-G’). The glands nonetheless invagianted and within

the invaginated portion of the glands Crumbs was localized apically. This suggests

that epithelial integrity and or polarity might be impaired if levels of Profilin are

imbalanced. Effects on junctional Armadillo in the absence of Profillin have been

described (TOWNSLEY and BIENZ 2000). In chic mutant embryos (either chic221 or

chic01320) at a stage when the first cells had just invaginated from the salivary gland

placode, cell shapes within the placode often appeared irregular compared to wild-

type, though Crumbs was still localized apically at this stage (compare Fig. 3 H, H’

and I). At later stages the salivary glands invaginated but were irregular in shape

and the placodal and surrounding epidermal cells on the surface of the embryo

appeared disrupted with absent or mislocalized Crumbs labelling (Fig. 3, K-L and O-

P at stage 12, M-N at stage 14, for comparison a matching wild-type epidermal scan

at stage 14 is shown in Q-Q’’). Other apical markers such as the spectraplakin Shot

and DE-Cadherin also appeared disrupted at these stages (Fig. 3V-V’’ for DE-

Cadherin and data not shown). Nonetheless, within the invaginated portion of the

glands Crumbs was localized apically, probably because early apical Crumbs

localization in the placodal cells was unperturbed. Thus, in contrast to the previous

report (CHANDRASEKARAN and BECKENDORF 2005) either elevation or disruption of

Chickadee/Profilin levels appeared to affect salivary gland invagination to some

extent. As Profilin has been shown to both promote actin polymerisation and

depolymerisation, depending on the context and tissue (YARMOLA and BUBB 2006),

an imbalance of Profilin levels (either lowered or increased) could affect critical

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cortical function of actin during cell shape changes required to allow the invagination

and/or the cell rearrangements on the surface of the embryo during invagination.

Rhomboid1 and EGF-receptor signalling are known to influence cell fate

decisions within the salivary gland primordium. The EGF-ligand Spitz is secreted

from the ventral midline cells with Rhomboid being the intramembrane protease

essential for its release (SHILO 2005). Spitz diffuses a few cell diameters laterally, to

induce the most ventral cells within the salivary gland placode to become duct cells,

whereas the other placodal cell become secretory cells (see scheme in Fig.4A). This

switch in fate transmitted by EGF is in part achieved through repression of the fkh

transcription factor. Fkh in turn represses three duct-specific genes, trachealess

(trh), dead ringer (dri) and serrate (ser) (HABERMAN et al. 2003; KUO et al. 1996).

Thus, by the end of embryogenesis rhomboid1 and other spitz group mutants have

salivary glands that are entirely composed of secretory cells and are completely

enclosed within the embryo without any ductal connection to the outside (Fig. 5 A-B’;

(KUO et al. 1996)).

Overexpression of rhomboid1 using EP(3)3704 in the glands led to the

formation of glands that were positioned too anteriorly with no obvious distinction in

shape between duct and secretory cells and no common duct-like structure formed

at stage 15 (Fig.4 C-C’’). An identical phenotype was observed when a rhomboid1

transgene under the control of the UAS promoter was expressed in the glands

(Fig.4 D-D’’). At earlier stages during the invagination process, when rhomboid1 was

overexpressed in the gland primordium using fkhGal4 and either the EP(3)3704 or

UAS-rhomboid1, aberrantly shaped lumena could be observed (Fig.4 E, E’) but most

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prominently a large ‘bulge’ of fkh-expressing ectopic cells seemed to arise between

the already invaginated secretory gland portions at the position where usually the

individual ducts would form (Fig.4 F, F’; similar ectopic cells could also be observed

in other experimental situations, see below). Though the analysis of rhomboid1

mutant embryos has shown that EGFR signalling is necessary to induce duct fate in

the most ventral cells of the placode (and thus loss of EGFR signalling leads to loss

of ducts, see Fig.5 A-B’), this could indicate that activation of EGFR signalling

throughout the placode was not sufficient to induce duct fate in all cells. To test this

hypothesis, we labelled embryos overexpressing rhomboid1 with markers for duct

(Eye gone, Eyg; JONES et al. 1998) and secretory (dCreb-A; ABRAMS and ANDREW

2005) gland fate and compared the expression to wild-type embryos. Both markers

labelled the salivary placode (Fig. 4, G and K), the glands at stage 14 (Fig. 4, H and

L) and stage 15 (Fig. 4, I and M) in a comparable pattern to wild-type placodes and

glands. In addition, the ‘bulge’ of potentially ectopic cells at the ventral surface that

was observed at stages 13-14 (bracket in Fig. 4L) strongly expressed the duct

marker Eyg, indicating that cells fated to become duct have overproliferated. At

stage 15-16, a large group of cells at the very anterior tip of the embryo that

completely failed to invaginate expressed Eyg (Fig. 4 M and M’’). These data

suggest that, at least when the EGFR pathway is ectopically activated throughout

the whole placode in a timeframe that mimicked the expression of fkh, this was not

enough to convert secretory cells into duct cells. To test whether this ‘failure in fate

conversion’ could be due to the timing of the overexpression, we also expressed

rhomboid1 under the control of armadilloGal4 (armGal4) throughout the whole

epidermis of the embryo and with expression starting at much earlier stages (Fig. 4

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N-P’’). The overactivation of EGFR signalling throughout the epidermis led to

embryos with varying degrees of overall affected morphology (in many cases head

involution failed, and general appearance of the epidermis was less organized

compared to wild-type, though epithelial integrity/polarity appeared unperturbed

judged by UAS-α-cateninGFP labelling that was also driven under the control of

armGal4). In these embryos dCreb-A and Eyg were expressed in the placode at

stage 11, though the dCReb-A expression domain appeared to extend beyond the

placode area into the more anterior hemisegments (Fig. 4 N,N’). Salivary gland

invagination was strongly affected in that only very short glands invaginated into the

embryo (see Fig. 4 O and P for stage 13 and 14, respectively). Nontheless, these

stubby glands expressed dCreb-A, the secretory fate marker, in the invaginated

portion of the glands (Fig. 4 O and P, arrows), and expressed Eyg in a few cells that

had invaginated but were still close to the surface of the embryo (Fig. 4 P’’). In

addition, a large ‘bulge’ of Eyg expressing cells could be found at the surface of the

embryo between the two invagination sides (Fig. 4 O, bracket), similar to what we

observed when rhomboid1 was overexpressed under the control of fkhGal4.

We also tested this hypothesis further by overexpressing additional

components of the EGF pathway in the salivary glands: a constitutively active

version of the EGFR, UAS-CA-EGFR, a secreted version of the ligand Spitz, UAS-

sspi, and the negative regulator Argos, UAS-argos. Overexpression of argos using

fkhGal4 led to a high proportion of glands that lost a ductal connection to the embryo

surface, similar to the rhomboid1 mutant embryos (Fig.5 A-D’’), though less

penetrant (which is probably due to timing and/or expression levels of the

transgene). When a secreted version of the EGFR ligand Spitz was expressed using

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fkhGal4 the phenotypes observed appeared very similar to the ones seen in the

rhomboid1 overexpressing embryos, namely ectopic cells and glands positioned too

anteriorly without discernable duct (Fig.5 K-L’). Overexpression of a constitutively

active form of the EGFR, UAS-CA-EGFR, in the salivary glands led to invagination

of cells with slightly aberrant shapes, and an invagination ‘hole’ that was too large.

This led to fully invaginated glands that had a too large and aberrantly shaped

lumen, though the individual and common ducts appeared normal (Fig.5 G-H’). The

ectopic ventral cells observed upon expression of rhomboid1 or secreted spitz in the

glands could be a result of overproliferation if EGFR signalling in the placodal cells

is not only working as a ‘fate switch’ but is also a mitogenic signal. We thus

analyzed the amount of cell division in the placode at stage 12-13 in embryos

overexpressing rhomboid1 or secreted spitz under the control of fkhGal4 compared

to wild-type placodes using an antibody against phosphorylated histone H3 (p-

HisH3), a chromatin mark of mitotic cells (WEI et al. 1999). In control embryos,

salivary placode cell nuclei at stage 12-13 did not contain nuclei showing p-HisH3

labelling (Fig. 5 M and P), whereas many placode cells overexpressing secreted

spitz (Fig. 5 N and Q) or rhomboid1 (Fig. 5 O and R) showed the p-HisH3 mark and

were thus actively dividing. Also, labelling of microtubules with GFP-EFGas2

revealed mitotic figures (Fig. 5O’ and O’’, dotted lines) and remnant spindle

midbodies (Fig. 5 O’ and O’’, arrows).

The results presented above suggest the following: firstly, that EGFR

signalling, though necessary for duct fate, is not sufficient to induce duct fate in all

salivary placode cells, even though absence of EGFR signalling turns all cells into

secretory cells. Secondly, an increase of EGFR signalling in the placode cells can

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induce excessive proliferation in a part of these cells, probably resulting in the

mishapen glands observed upon rhomboid1 or spitz overexpression. Thirdly,

through modulation of fkh levels and downstream components within the placode,

EGFR signalling might also impinge on the cell shape changes that invaginating

cells undergo.

Egalitarian (Egl) and BicaudalD (BicD) are two proteins that act together with

cytoplasmic Dynein in the localization of mRNAs in Drosophila embryos and the

oocyte, with Egl interacting directly with Dynein light chain (BULLOCK and ISH-

HOROWICZ 2001; NAVARRO et al. 2004). Overexpression of egl using EP(2)938 led to

salivary glands that were C-shaped or shortened (Fig.6 B and C). Shortened glands

could also be observed when egl was expressed in the glands using a UAS-egl

construct (Fig.6 D and D’). GFP-positive cells could be observed that appeared to

lose contact with the gland (arrow in Fig.6 D). Because both BicD and Egl have

essential functions during oogenesis an analysis of egl or BicD null embryos is not

possible. We therefore analyzed embryos from mothers carrying two hypomorphic

alleles of egl that were mated to heterozygous fathers (see Materials and Methods

for the exact genotypes). Embryos with reduced Egl function often showed a

disrupted epidermis, with large patches that appeared to completely lack apical

Crumbs labelling (Fig.6 F,F’ compared to Fig.6 G, G’). This phenotype was variable,

though, and an example of an embryo with less disrupted epidermis is shown in

Fig.6H. Also, during later stages of invagination at stage 13, the placode area was

often disrupted and lacked apical Crumbs (Fig.6 K). Salivary gland morphogenesis

was disrupted in egl mutant embryos in that the cells of the placode often did not

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change their apices in a coordinated way (though crumbs still accumulated apically

in the placode, see Fig. 6H’), the invagination hole appeared too large and extended

(Fig.6 H) and the invaginated portion of the glands often had an irregular shape

(Fig.6 I and K’). In the invaginated portion of a gland, Crumbs was not concentrated

near the apical cell junctions as in the wild-type, where this accumulation appears

as a honeycomb lattice, (compare Fig.6 L’ and Fig.6 M’). Instead, Crumbs was

delocalized all over the apical surface and large accumulations could also be found

intracellularly (arrow in Fig.6 L’). What could be the mechanism leading to a loss of

apical surface identity or constituents? Egl and BicD together with Dynein act as

minus-end directed microtubule motors, and as in most epithelial cells the minus

ends of microtubules are located near the apical surface in the salivary glands

(MYAT and ANDREW 2002). hairy mRNA is one of the best understood cargoes of Egl

and BicD mediated transport (BULLOCK et al. 2006; BULLOCK et al. 2003), and Hairy

has been shown to be important for the regulation of apical membrane growth

during salivary gland formation, in part through modulation of Crumbs (MYAT and

ANDREW 2002). Thus, affecting hairy transcript localization through lowered levels of

Egl and BicD could in turn affect the maintenance of apical membrane identity in the

secretory cells. Alternatively, recent reports have shown that crumbs mRNA itself,

and also the RNA of the Crumbs-associated protein Stardust (Std), are apically

localized and this apical localization is important for function (HORNE-BADOVINAC and

BILDER 2008; LI et al. 2008). Thus, if crumbs mRNA localization were dependent

upon Egl and BicD, then reduction of Egl and BicD would result in loss of functional

crumbs at the apical surface, leading to loss of epithelial characteristics. The apical

localization at least of std mRNA appears developmentally regulated in the embryo

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(HORNE-BADOVINAC and BILDER 2008). Thus, it is possible to envisage that salivary

gland cell apical maintenance is Egl and BicD dependent and especially sensitive to

levels of Egl and thus Crumbs in comparison to other epithelial tissues at the same

stage. We are currently investigating this issue in more detail.

A gene endogenously expressed in glands identified through antisense

expression:

Bitesize (Btsz) is the sole Drosophila synaptotagmin-like protein. Its mRNA

is expressed in the salivary glands and also other epithelial tissues, with a strong

apical enrichment (SERANO and RUBIN 2003). Btsz has recently been shown to

control the organization of actin at adherens junctions in early embryos, though it

might be dispensable in adult fly epithelia (PILOT et al. 2006). Recruitment of Btsz in

early embryos to the apical junctional region is not dependent on E-Cadherin but on

PIP2 (phosphatidylinositol-(4,5)-bisphosphate) and Par-3/Bazooka, a protein of the

Par-3/Par-6/aPKC apical complex (PILOT et al. 2006). Two mutant btsz alleles have

been described, btszK13-4 that deletes a portion of the N-terminus of some Btsz

protein isoforms, and btszJ5-2 that introduces a stop codon due to a frameshift in the

N-terminal portion of btsz (SERANO and RUBIN 2003). Expression driven by fkhGal4

from the EP-element identified in our screen, EP(3)3567, should lead to production

of an antisense RNA to most of the btsz coding sequence and thus could

downregulate endogenous btsz mRNA levels (see scheme in Fig. 7A and

Supplementary Figure 2). In embryos where EP(3)3567 is driven by fkhGal4 at

stage 13, when most secretory cells have invaginated from the placode, the

epidermis in the region where the antisense RNA was expressed was disrupted and

21

had lost apical Crumbs accumulation (Fig.7 B and B’). The glands themselves

showed an irregular lumen (Fig.7 C and C’). btszK13-4 mutant embryos that manage

to cellularize and complete gastrulation showed a somewhat disrupted epidermis,

with loss of apical Crumbs accumulation in patches, at later stages (see Fig.7 D for

a stage 13 and Fig.7 F for a stage 14 embryo). Nonetheless, many salivary gland

placode cells still showed enhanced Crumbs labelling, though shapes of apical

circumferences of invaginating cells were irregular (Fig.7 D-D’, compare to wild-type

in Fig.7 E-E’). Also the apical accumulation of the fly spectraplakin protein Shot

could not be observed in btszK13-4 embryos in contrast to wild-type (Fig. 7 D’’ versus

E’’). At stage 14 the secretory portion of the glands in btszK13-4 embryos was often

found to lose apical localization of Crumbs (and also show reduced apical actin

enrichment; Fig.7 G-G’’). Similar to what we observed upon expression of

EP(3)3567 with fkhGal4, the epidermis in the region where the placodal cells were

located previously was disrupted and lost apical Crumbs and also DE-Cadherin

labelling completely (Fig.7 H versus wild-type in Fig.7 L; Fig.7 M). The disruption of

the epidermis and failure in proper apical localization of Crumbs is to some extent

reminiscent of the phenotypes observed in egl mutant embryos. As btsz mRNA is

another RNA that is localized apically in various epithelial cells (and the localization

signal has been identified, SERANO and RUBIN 2003) one could speculate that its

localization could also be dependent on Egl and BicD, thus explaining some overlap

in the phenotypes.

A gene not endogenously expressed in the glands identified through

overexpression:

22

Capricious (Caps) belongs to the class of leucine-reach-repeat (LRR)

transmembrane proteins, together with its close paralogue Tartan (Trn). Both

proteins have been implicated in the formation of compartments of cells with

different affinities in the wing disc (MILAN et al. 2001), modulation of epithelial

integrity within the wing disc (MAO et al. 2008), correct targeting of a subset of

photoreceptor axons to the correct layer within the optic lobe (SHINZA-KAMEDA et al.

2006), and also joining of tracheal branches over segment boundaries (KRAUSE et

al. 2006). One study also showed that in tissue culture Capricious and Tartan are

able to mediate homophilic cell adhesion, a molecular function that could explain

their roles discussed above (SHINZA-KAMEDA et al. 2006). Overexpression of

capricious using EP(3)552 led to glands with an enlarged lumen and very aberrant

shapes at stage 15 (Fig. 8 B and C). The same phenotype was observed when a

transgene of capricious was expressed under UAS control (Fig.8 D-E’). During early

stages of invagination, the invagination hole appeared enlarged compared to wild-

type and extended along the anterior-posterior axis (compare Fig.8 F-G’, and Fig.8

H and H’), suggesting problems in the shape of invagination cells and the order of

invagination. This disorganization could at later stages lead to the aberrant shapes

of the secretory part of the glands observed. We then analyzed capricious and

tartan single and capricious tartan double mutant embryos (MAO et al. 2008), as

some previous studies have indicated redundancy between both molecules in some

tissues (MAO et al. 2008). In all mutant situations invaginating glands often showed

irregular lumens (Fig.8 I-M), indicating that both proteins might work together during

salivary gland invagination. We next analyzed if and where capricious and tartan are

expressed during salivary gland morphogenesis using P-element insertions into

23

each gene that carry a lacZ gene leading to β-galactosidase expression under the

endogenous expression control of each of the genes. Capricious was expressed in

the embryo in the region of the salivary glands from stage 12-15, but appeared to be

mostly excluded from the salivary glands themselves, though it was always

expressed in cells in close contact with the glands (Fig.8 N-S). In contrast, tartan

was expressed strongly in the salivary glands from placode stage on (Fig.8 T-U).

Similar expression patterns could also be observed in in situ hybridization for

capricious and tartan mRNAs (see supplementary Figures 33 and 4). Thus, in

analogy to the situation in the developing trachae where both protein are expressed

in ‘complementary’ tissues to allow for proper dorsal branch fusion (KRAUSE et al.

2006), the reciprocal expression of capricious and tartan in and around the salivary

glands could play a part in the correct invagination and later positioning of the

glands with respect to the surrounding tissues.

24

DISCUSSION

Here we show that a gain-of-function screen looking for factors affecting a process

of epithelial morphogenesis, the formation of the salivary glands in the Drosophila

embryos, was efficient in identifying a range of known and new players. The screen

was designed to identify genes that are endogenously expressed in the glands and

where overexpression or antisense expression by an EP element interfered with

endogenous function. The screen could also identify genes not endogenously

expressed in the glands but with an apparent overexpression phenotype that

uncovered a potential function for this gene in cell shape or cytoskeletal regulation

elsewhere. Genes falling into all of these classes have been identified in the screen.

We show that factors with a variety of proposed functions can affect salivary gland

invagination, from cytoskeletal components, via signalling factors (some of which

impinge on the cytoskeleton themselves), to microRNAs and novel uncharacterized

proteins. Most of the factors identified in this screen should affect salivary gland

morphogenesis in a gland-autonomous fashion due to the restriction of

overexpression to the salivary glands only, the only exception being the

overexpression of secreted signalling factors.

We could observe a variety of phenotypes from aberrantly-shaped glands to

irregular lumena and wrongly positioned glands. In half of the cases, upon

overexpression of a gene (or in a few cases potentially an antisense RNA) several

different phenotypes could be observed, as opposed to a single dominant

phenotype that was found in the other half. At the level of detail at which we

analyzed the phenotypes (GFP-markers of cell shape or microtubules plus

25

phalloidin-labelling of actin), these seemed to fall into a limited number of classes,

suggesting that several different perturbances of the system might lead to similar

phenotypes. We also found no case that consistently led to a complete failure in

salivary gland invagination. This is not completely surprising, as dominant effects or

knock-downs that we would expect to see in our screen might not perturb the

system enough to lead to a complete failure in all aspects of the cell shape changes

required for the invagination. Also, when analyzing various mutants in the initial

follow-up of a subset of the hits identified, even in situations where the embryonic

epidermis seemed to be very disrupted and cell shapes of invaginating cells were

highly irregular as for instance in btsz or egl mutants, the glands nonetheless

managed to invaginate. These observations suggest that there is a strong ‘drive’ for

the invagination of the cells of the salivary gland primordium, with many different

factors contributing at the effector level. Elimination or perturbance of only one of

these factors will not prevent invagination completely, but will rather lead to a slightly

disordered invagination process that in the end might result in the aberrant shapes

and phenotypes we observed. This situation appears similar to the process of

mesoderm invagination during gastrulation in the Drosophila embryo. Many factors

contribute to this process, but loss of none apart from the most upstream

transcription factor initiating the whole mesoderm invagination program, twist, will

abolish invagination completely. In all other downstream mutants analyzed the

mesoderm will nonetheless manage to invaginate, albeit in an uncoordinated and/or

delayed fashion (LEPTIN 2005). Similarly, during salivary gland invagiantion, fkh

appears to be the most upstream transcription factor initiating the ‘invagination

program’ for both the secretory and the ductal part of the glands. In the absence of

26

Fkh, invagination fails completely (MYAT and ANDREW 2000a). Several direct targets

of Fkh have been identified, including the transcription factors senseless

(CHANDRASEKARAN and BECKENDORF 2003) and sage (ABRAMS et al. 2006),

PH4αSG2, a prolyl-4-hydroxylase (ABRAMS et al. 2006), and also crebA, which in

turns control the expression of secretory genes in the glands (ABRAMS and ANDREW

2005).

Only one recent study has so far addressed direct targets of fkh in a genome-

wide manner (LIU and LEHMANN 2008). In this study, whole genome expression

levels were compared between control pupae and pupae with forced expression of

fkh. At the beginning of pupariation fkh controls both the expression of the salivary

gland secretion proteins (ROTH et al. 1999) and also controls the cell death that

occurs during pupariation in this tissue (MYAT and ANDREW 2000a). Downstream

targets of fkh identified in this study included cell death genes, genes involved in

autophagy, phospholipid metabolism, glucose and fatty acid metabolism and

hormone-dependent signalling pathways, but also others. These other factors

regulated by fkh included several proteins that we also identified in our screen:

capricious, bitesize, gliotactin, ptpmeg, rhomboid1, and spitz. This overlap of fkh-

dependent factors and genes identified by us, that are also potentially fkh-

dependent, at different stages of development, i.e. embryo and pupa, could suggest

that these overlapping factors are regulated by fkh independent of stage specific co-

factors.

The set of genes identified in our screen encode proteins with a wide range of

potential functions: cytoskeleton or cytoskeleton-associated, signalling, nuclear or

transcription factor, protein synthesis and degradation, membrane traffic, cell

27

surface and extracellular, enzymes, mitosis-meiosis-germline, and also

uncharacterized genes and microRNAs. Nonetheless, many of these have been

implicated in some aspect of epithelial morphogenetic function, some even within

the salivary glands themselves. Thus, we are confident that many of the factors

identified in this gain-of-function screen will turn out to have a function in epithelial

morphogenesis. The initial folllow-up of the subset of genes described in detail

above also confirms that gain-of-function phenotypes can point to new factors

involved in a process, but can also reveal new aspects of a function of a protein that

were not previously appreciated, as in the case of rhomboid1.

The overexpression phenotype of rhomboid1 in the salivary glands showed

an intriguing phenotype; ectopic EGFR signalling throughout the part of the

primordium that will constitute the secretory part of the gland does not simply induce

these cells to switch to ductal fate. Instead it suggests that either other permissive

factors are expressed in the duct primordium independent of EGFR signalling that

are absent from the secretory primordium, or further inhibitory factors are at work in

the secretory primordium in addition to EGF-induced fkh that prevent ductal fate.

The timing of the overexpression of rhomboid1 in the screen, using part of the fkh

promoter in the fkhGal4 line to drive the expression of the EP elements, did not

seem to be crucial to the fate decision, as expression of rhomboid1 throughout the

whole epidermis using and ealier Gal4-driver (armGal4) still lead to invagination of

glands with identifiable secretory and ductal cells (Fig. 4, N-P). Thus it does not

appear that the cells in the placode are responsive to the EGFR signal in terms of

fate assignment only in a narrow time window, but supports the notion that other

28

factors are involved. It will be interesting to determine in the future what these

factors are.

The analysis of caps and tartan mutant phenotypes suggests a role for these

genes in salivary gland morphogenesis. The molecular function of both Caps and

Trn proteins is still unclear. They appear important to mediate interaction and

disctinctiveness between groups of cells: neurons finding appropriate targets in the

brain (SHINZA-KAMEDA et al. 2006; SHISHIDO et al. 1998), separation of ventral and

dorsal compartment cells in the wing disc (MILAN et al. 2001), and tracheal

morphogenesis across segment boundaries (KRAUSE et al. 2006). Caps and Trn

have been suggested to act as homophilic or heterophilic adhesion receptor, or

serve another unidentified function during adhesion. Our results indicate that

salivary gland morphogenesis might be a useful system to address their molecular

function in more detail. It is also interesting to note that not only Caps and Trn, but

also Slit and the protein encoded by CG14351, which were also both identified as

hits in the screen, belong to the family of LRR proteins, suggesting a general role for

this class of surface receptors in salivary gland morphogenesis.

Another gene identified in the screen with a highly penetrant phenotype

(severely shortened glands) is TNF-receptor-associated factor-4 (traf-4, previously

annotated as traf-1 in Flybase). Traf-4 has previously been shown to induce

apoptosis via activation of JNK-kinase when overexpressed in other tissues

(KURANAGA et al. 2002). Traf-4 has also been linked to the Ste20 kinase Mishapen

(another gene identified in our screen) which in turn has been shown to be important

for coordinated cell shape changes occurring for instance during dorsal closure in

the fly embryo and epiboly in the zebrafish embryo (KOPPEN et al. 2006). It also

29

appears to have a role in mesoderm invagination (Maria Leptin; personal

communication). Overexpression of traf-4 using EP(2)578 led to a severe reduction

in the number of cells in the salivary glands at embryonic stage 15 (when counting

cell numbers in fluorescence images taken through the middle of wild-type glands,

these had 39.9 +/- 3.9 cells (n= 40) around the perimeter of the gland, whereas traf-

4 overexpressing glands had 18.7 +/- 5.0 cells (n=45) around the perimeter). We

have not directly tested that the missing cells have died through induction of

apoptosis, but would expect this to be the case in agreement with the earlier

studies. To address whether traf-4 has a function linked to mishapen and cell shape

changes in the glands we analyzed fly embryos lacking Traf-4 (this mutant was a

kind gift of Maria Leptin) for any problems in the early cell shape changes occurring

during salivary gland invagination, but could not find any strong defects (data not

shown). Thus, despite many similarities between the epithelial morphogenetic

processes of mesoderm invagination (which also starts with the invagination of an

epithelial sheet) and salivary gland invagination, downstream effectors vary between

the two systems.

Another interesting group of hits identified in the screen are the EP-elements

potentially driving overexpression of the mir-310 microRNA cluster. This cluster

contains the microRNA genes mir-310, mir-311, mir-312, and mir-313.

Overexpression of the cluster from three different EP-elements located just

upstream of the cluster, EP(2)2536, EP(2)2586 and EP(2)2587, led in each case to

glands with widened and irregular lumena, though with varying penetrance (data not

shown). Antisense-mediated depletion of each microRNA from this cluster has

previously been shown to perturb dorsal closure and head involution in the embryo,

30

indicating that the inhibition of downstream targets of this cluster might be important

for various epithelial morphogenetic events (LEAMAN et al. 2005). Thus, the

microRNAs in this cluster could be important to regulate targets that require

downregulation to facilitate invagination during salivary gland morphogenesis. Two

other microRNAs have been shown to be expressed in the salivary gland in the

embryo, mir-8 and mir-375, with mir-8 showing a dynamic expression pattern

(ABOOBAKER et al. 2005), expression patterns for the mir-310 cluster have not been

analyzed yet. This data together with our screen results suggests that microRNA-

dependent control of gene expression might be an important factor in salivary gland

morphogenesis.

Thus in summary the gain-of-function screen for factors affecting cell shape

during salivary gland morphogenesis in the Drosophila embryo presented here was

successful in identifying a range of candidates. These candidates represent on the

one hand genes that are endogenously expressed in the glands and thus are likely

to be serve a role during salivary gland morphogenesis/tubulogenesis. On the other

hand we identified genes that are not endogenously expressed in the glands but

nonetheless interfered with their invagination, potentially through effects on cell

shape or the cytoskeleton. These genes might therefore be important for the

regulation of cell shape either in other tissues. It will be interesting to determine in

the future which of the candidate genes of the first class serve a function only in the

salivary glands, and which are required for tubulogenesis events in general, also in

other species. For the second group of candidates an analysis of their role during

cell shape changes in other morphogenetic events will be key to understanding how

they affected salivary gland morphogenesis in our screen. As the screen identified

31

several uncharacterized genes whose expression gave strong phenotypes in the

glands and that have close orthologues in mammals, the salivary glands appear to

be a good model system to analyze the function of such genes. Finally, our follow-

up investigation of a selected set of candidate genes through loss-of-function

mutants demonstrates that the combination of functional screening and phenotypic

loss-of-function analysis provides a useful approach to identify downstream

effectors in a morphogenetic process.

32

ACKNOWLEDGEMENTS

The authors would like to thank Nick Brown, Matthew Freeman, Simon Bullock,

Deborah Andrew, Maria Leptin and the Bloomington and Szeged Stock Centres for

fly stocks; Matthew Freeman, Simon Bullock, Sarah Bray, Deborah Andrew and the

Developmental Studies Hybridoma Bank at the University of Iowa for antibodies;

Maria Leptin for communication of results prior to publication; Nick Brown for use of

his confocal microscope; Sean Munro and Nick Brown for helpful comments on the

manuscript. This work was supported by the Biotechnology and Biological Sciences

Research Council [grant number BB/B501798/1] and the Royal Society.

33

MATERIALS & METHODS

Screen Design and Fly Husbandry

‘Marker’-lines: fkhGal4 (HENDERSON and ANDREW 2000; ZHOU et al. 2001) was

recombined on the third chromosome with a UAS-construct containing GFP fused to

the N-terminus of the EF-Gas2 region of Shot (SUBRAMANIAN et al. 2003) or the

membrane targeting domain of src fused to GFP (KALTSCHMIDT et al. 2000). One or

the other of these marker lines were crossed to 1001 EP lines from the Rørth

collection obtained from the stock centres in Szeged (second and third

chromosomes; http://expbio.bio.u-szeged.hu/fly/index.php) and Bloomington (third

chromosome; http://flystocks.bio.indiana.edu/). The fkhGal4 insertion was a gift from

Deborah Andrew, the SrcGFP from Nick Brown. UAS-chickadee was from Lynn

Cooley. rho[PΔ5], argos[lΔ7], flb[ik35], UAS-argos, UAS-CA-EGFR, UAS-sspi, UAS-

caps, caps[PB1], trn[28.4], caps[Del1] trn[28.4] alleles were gifts from Matthew

Freeman; egl[3e], egl[PR29], BicD[HA40], b BicD[18a], dp b Df(2L)TW119 and UAS-

egl were gifts from Simon Bullock (the UAS-egl transgene leads to an approximate

3-fold increase in levels; Simon Bullock, personal communication). To analyze egl

mutant embryos, egl[3e]/egl[WU50] females were mated to egl[PR29]/+ males, and

to analyze BicD mutant embryos BicD[HA40]/+; b BicD[18a]/ dp b Df(2L)TW119

mothers were mated to BicD[18a]/CyO males. In the detailed analyses (apart from

in the case of egl and BicD mutant embryos), mutant embryos were identified by the

absence of green balancer (balancer chromosomes used were CyO Kr::GFP, TM3

Sb Ser twi-gal4 UAS-2x eGFP, and TM6b Tb Sb df-Gal4 UAS-YFP). All other stocks

34

used were from the Bloomington Stock Center. Crosses were maintained at 25ºC on

cornmeal food, embryos were collected on apple or grape juice-agar plates.

EP lines were determined to be heterozygous or homozygous for the EP insertion.

In the absence of visible balancer chromosomes, lines were assumed to be

homozygous. Homozygous lines were crossed to a homozygous driver line and

embryos were collected over night on apple or grape juice plates with yeast paste.

20 embryos between stage 10 and 13 and 20 embryos between stage 13 and 15

(an “early” and a “late” sample) were scored in live mounts in halocarbon oil

(Halocarbon Oil 27, Sigma) after dechorionation in 50% bleach. In heterozygous

balanced lines, 40 embryos in each of the early and late group were scored,

assuming equal fertilisation and survival from both genotypes through the end of

embryogenesis. Lines with ≥ 20% salivary gland defects, or with potential defects

that would require quantitative analysis (i.e. changes in length), were subjected to

second pass screening. In the second pass, embryos were collected as above, fixed

in 2:1 heptane:4% formaldehyde in PBS and stained with rhodamine phalloidin. A

larger number of embryos was scored from these collections (average >80). First

pass hits were also crossed to w f flies, embryos collected and stained with

rhodamine phalloidin as above to check for dominant positional effects of the EP

insertion.

Immunohistochemistry, Widefield Fluorescence and Confocal Analysis

Embryos were collected on grape-juice plates and processed for

immunofluorescence using standard procedures. Briefly, embryos were

dechorionated in 50% bleach, fixed in 4% formaldehyde and stained with phalloidin

35

or primary and secondary antibodies in PBT (PBS plus 0.5% bovine serum albumin

and 0.3% Triton X-100). Crumbs and DE-Cadherin antibodies were obtained from

the Developmental Studies Hybridoma Bank at the University of Iowa; the Shot

antibody was raised in our lab and is identical in design to the one described in

(STRUMPF and VOLK 1998); the anti-dCrebA antibody was from Deborah Andrew

(ANDREW et al. 1997); the anti-phospho-histone H3 and anti-GFP antibodies were

from abcam (UK). Secondary antibodies used were Alexa Fluor 488-coupled

(Molecular Probes) and Cy3- and Cy5-coupled (Jackson ImmunoResearch

Laboratories Inc.), rhodamine-phalloidin was from Molecular Probes. Samples were

embedded in Vectashield (Vector Laboratories). Widefield fluorescence images

documenting the screen results were obtained on a Leica DMR (equipped with a

MicroFire camera, Optronics), a Zeiss Axioplan 2 (equipped with a Princeton

Instruments camera) and a Zeiss Axioskop Mot 2 (equipped with a Jenoptik C14

camera), using PictureFrame, Metamorph and Openlab software, respectively.

Confocal images were obtained using an Olympus Fluoview 1000. Confocal laser,

iris and amplification settings in experiments comparing intensities of labelling were

set to identical values. Widefield fluorescence and confocal images were assembled

in Adobe Photoshop, confocal z-stacks and z-stack projections were assembled in

Image J.

In Situ Hybridization

In situ hybridization of whole-mount embryos was performed essentially as

described by (TAUTZ and PFEIFLE 1989). To combine the in situ protocol with

immunohistochemistry for GFP, the anti-GFP antibody was incubated together with

36

the anti-DIG antibody, followed by fluorescent secondary antibody incubation to

reveal the GFP after the BCIP/NBT colour reaction. Images were obtained on a

Leica DMR (equipped with a MicroFire camera, Optronics) and composites were

assembled using Adobe Photoshop.

The following primers were used to generate in situ probes: chic 5’

TTTCCATCTACGAGGATCCC, chic 3’ ATTTCGTTCAAAGCTGAGGAC; caps 5’

CGGGCAATTACCATGTCGTTG, caps 3’ GATGTGGCTGATGCGATTCTG; trn 5’

GTGGGCATCTGGTGCATTTTG, trn 3’ GATAAAGGATGCGCAACTGGG; for the

btsz probe the cDNA clone AY229970 was used to transcribe antisense and sense

probes.

Statistics

We determine the ‘base rate’ of salivary gland defects observable in our

experimental stocks by counting defects in the genotype: +/+; +/CyO; fkhGal4::UAS-

GFPmarker/+. The base rate in this genetic background was determined as 4.3%

(n=748). A similar base rate was obtained in a genetic background where the CyO

balancer chromosome was replaced by a GFP-marked chromosome:

+/+;+/btlGal4::UAS-GFP; fkhGal4::UAS-GFPmarker/+; the base rate was 4.4%

(n=878). Thus, we exclude any effect at least of a CyO balance chromosome

present on salivary gland morphogenesis.

To set a cut-off level for the rate of affected salivary glands counted in each

experiment, above which we determined EP-elements driven by fkhGal4 affect the

salivary gland morphogenesis, we chose an arbitrary 20% defects cut-off for the first

pass analysis. This yielded 187 EP lines (=18.6% of the total lines screened) to be

37

re-screened in the second pass analysis, yielding 51 confirmed insertions overall.

This equals ~5% of the total number of EP lines screened, which also equals two

standard deviations form the mean of a normal distributed sample, indicating that

we set our cut-off at a sensible level.

38

Table 1. Genes identified in the gain-of-function screen.

This table lists all the genes identified in the screen, sorted according to their proposed function.

EP number Cytology Gene affected Effect, Location in Gene, Direction?

Penetrance of Phenotypes *

Phenotype in Salivary Glands (fkhGal4)

Function or Mutant Phenotype, Known Function in Flies or Glands

Cytoskeleton & Cytoskeleton-associated

EP(2)570 2L (26B4) CG13993 (actin/tubulin protein folding)

overexpression, 5’ end of gene

strong variable no mutant available

EP(2)713 2L (26B1) chickadee (profilin) overexpression, in 5’ region of gene upstream of most CDS

strong variable lethal, Tec29 chic double mutants show salivary gland phenotype (CHANDRASEKARAN and BECKENDORF 2005)

EP(2)1011 2L (26B1) chickadee (profilin) antisense to 5’ 1kb of gene (or overexpression of eIF4a, 1.8kb downstream)

strong hooks, shepherd’s crook

lethal, Tec29 chic double mutants show salivary gland phenotype (CHANDRASEKARAN and BECKENDORF 2005)

EP(2)938 2R (59F7) egalitarian (dynactin-associated)

overexpression, directly 5’ of gene

strong variable female sterile, lethal, microtubule-based transport

EP(2)2047 2R (57E5) syndecan inserted in 5’ end of sdc wrong strand,

strong variable lethal, works in conjunction with Slit

39

could drive antisense to 6kb out of the 90kb sdc locus or overexpress sara 7kb downstream

(STEIGEMANN et al. 2004)

EP(3)3567 3R (88D5) bitesize (synaptotagmin-like protein)

middle of gene, could drive antisense to most isoforms

weak variable lethal, actin organization at adherens junctions (PILOT et al. 2006)

Signalling

EP(2)578 2L (24E1) traf-4 (TNF-receptor-associated factor 4, previously called Traf-1 in flies)

overexpression, middle of gene, upstream of most CDS

very strong less cells in glands, overexpression reportedly induces apoptosis (KURANAGA et al. 2002)

larval lethal (KURANAGA et al. 2002)

EP(2)2167 2L (29A1) btk29/tec29 (Btk family kinase)

3’ end of gene wrong strand, could drive antisense to >30kb of 40kb btk29A locus

weak variable lethal, Tec29 is important for sal. gland invagination (CHANDRASEKARAN and BECKENDORF 2005)

EP(2)1173 2L (37E1) ranGAP overexpression, middle of gene but upstream of CDS

strong hook, crook viable, possible link between nuclear transport, actin and profilin (MINAKHINA et al. 2005)

EP(2)2158 2L (37D2) doughnut on 2 (RYK family receptor tyrosine

overexpression weak variable important for salivary gland positioning as is drl another RYK

40

kinase) (HARRIS and BECKENDORF 2007)

EP(3)3542 3L (61C1) ptpmeg (tyrosine phosphatase) or mthl9 (G-protein coupled receptor)

overexpression of ptpmeg or antisense of mthl9 (intronic to ptpmeg)

strong budding viable, Ptpmeg is FERM domain protein

EP(3)3704 3L (62A2) rhomboid1 (EGF signalling, intramembrane protease)

overexpression very strong aberrant duct morphogenesis, potentially due to overproliferation (see Figs 4 and 5)

mutations in rho or spitz lead to transformation of duct cells into secretory cells (KUO et al. 1996)

EP(2)2201 2L (37F2) spitz (secreted EGF ligand)

inserted into middle of spi, could drive antisense to 4kb of some spi mRNAs, or overexpress msb1l 5kb downstream

weak variable mutations in rho or spitz lead to transformation of duct cells into secretory cells (KUO et al. 1996)

EP(3)549 3L (62E7) misshapen (Ste20 kinase)

overexpression, directly 5’ of gene

weak too wide and lumpy lumen

lethal, linked to nuclear movement via BicD (HOUALLA et al. 2005) and cell shape changes during morphogenesis (KOPPEN et al. 2006)

Nucleus, Transcription (Factors)

EP(2)2176 2R (48E4) Smd3 (snRNP, splicing)

overexpression, 5’ of gene

strong hooks lethal (SCHENKEL et al. 2002)

EP(2)474 2L (21B5) kismet (helicase) overexpression, in 5’ region of gene upstream of all CDS

weak too wide lumen lethal, segment specification (DAUBRESSE et al. 1999)

41

EP(2)993 2R (50E1) combgap (zinc-finger protein)

inserted into 5’ end of CG30096 wrong strand, antisense to cg 2kb away

very strong variable lethal, hedgehog signalling in leg patterning (SVENDSEN et al. 2000)

EP(3)486 3L (75E1) ftz-f1 (ftz-transcription factor1)

inserted into locus, should overexpress longer isoform

weak variable lethal (FLORENCE et al. 1997)

EP(3)711 3L (64E8) bre-1 (nuclear factor downstream of Notch)

overexpression, directly 5’ of gene

weak variable lethal (BRAY et al. 2005)

Protein Synthesis and Degradation

EP(2)463 2R (47F7) Tapδ (translocon-associated protein δ)

overexpression, 5’ end of gene

weak variable lethal, downstream of dCreb-A in the glands (ABRAMS and ANDREW 2005)

EP(2)2063 2L (37B7) nedd8 (regulation of proteolysis)

overexpression, 5’ end of gene

weak butterfly lethal, ubiquitin-like, cooperates with cullin3 (ZHU et al. 2005)

EP(2)1187 2L (33C1) CG5317 (ribosomal subunit)

overexpression, inserted in 5’ end of JhI-21, wrong strand, should drive CG5317 400bp downstream

strong shepherd’s crook

n.d.

Membrane Traffic

EP(2)2028 2R (48F8)

garz (arf-GEF, GBF1)

overexpression, 5’ end of gene

weak severe hooks ER to Golgi trafficking in mammals (SZUL et al. 2007)

EP(2)2313 2L (35F1) syntaxin5 (SNARE protein)

overexpression, directly 5’ of gene

weak degenerating glands

lethal, membrane fusion, cytokinesis (XU

42

et al. 2002)

Cell Surface & Extracellular

EP(2)827 2R (58D4) CG3624 (Ig domain protein)

overexpression, directly 5’ of gene

strong variable n.d.

EP(2)937 2R (52D1) slit (axon guidance receptor)

middle of gene, wrong strand, antisense to 20kb of 50kb gene?

strong hooks lethal, slit has been shown to be involved in salivary gland positioning (KOLESNIKOV and BECKENDORF 2005)

EP(2)2120 2L (22A3) CG14351 (LRR and Ig domain transmembrane protein)

overexpression, directly 5’ of gene

weak variable n.d., BLAST shows similarity to Slit

EP(2)2463 2L (35D4) gliotactin (transmembrane protein of septate junctions)

overexpression, directly 5’ of gene

weak variable lethal, important for tube size control in trachae (PAUL et al. 2003)

EP(3)552 3L (70A3) capricious (transmembrane LRR protein)

overexpression, directly 5’ of gene

very strong bizarrely branching and budding lumen

lethal (SHISHIDO et al. 1998)

Enzymes

EP(2)2199 2R (51B1) tout velu (glucosaminyl-transferase)

inserted in intron of both ttv and lamC (which is intronic to ttv), could overexpress ~10kb of 60kb ttv (about 50% of CDS) or

weak variable lethal, mutans disrupt hh, wnt and dpp sigalling (BORNEMANN et al. 2004)

43

1.1kb antisense to LamC

EP(2)1157 2R (59B6) CG9849 (potential protease of the subtilase family)

inserted into 5’ end of CG3800, wrong strand, overexpression of CG9849 600bp away

weak variable n.d.

EP(3)3639 3L (65A10)

CG10163 (phospholipase A1)

inserted 5’ of Best2, wrong strand, could drive antisense to CG10163 800bp away

weak too large and irregular lumen

n.d.

Mitosis, Meiosis, Germline

EP(2)812 2L (35C1)

vasa or vig (vasa intronic gene)

in 3’ region of vig which is intronic to vasa, would overexpress 3’ 1kb of vig or antisense to vasa

weak lumpy lumen vasa: lethal, germ cell determination/ vig: n.d.

EP(3)341 3R (82D2)

tacc (centrosomal protein)

middle of gene, could drive antisense to all large isoforms of tacc

weak variable lethal (BARROS et al. 2005)

Other

EP(2)2356 2R (57A6) mir-310/-313 cluster

overexpression, inserted 200bp 5’ of cluster

very strong to wide irregular lumen

disruption of mir-310 cluster affects dorsal closure (LEAMAN et al. 2005)

44

EP(2)2586 2R (57A6) mir-310/-313 cluster

overexpression, inserted 100bp 5’ of cluster

strong irregular lumen disruption of mir-310 cluster affects dorsal closure (LEAMAN et al. 2005)

EP(2)2587 2R (57A6) mir-310/-313 cluster

overexpression, inserted 100bp 5’ of cluster

weak irregular lumen disruption of mir-310 cluster affects dorsal closure (LEAMAN et al. 2005)

EP(2)1221 2L (27F4) mir-275, mir-305 overexpression, 2kb upstream of genes

strong shepherd’s crook

n.d.

EP(2)2083 2R (45F1) CG1888 >6kb away, overexpression

weak variable n.d.

EP(2)1163 2L (33E4) vir-1 (virus induced RNA 1)

overexpression, directly 5’ of gene

weak budding n.d.

EP(2)1239 2L (25F5) CG14005 or CG7239

inserted in 5’ end of CG9171 wrong strand, antisense to CG14005 300bp downstream or overexpression of CG7239 2kb downstream

weak variable n.d., both CG14005 and CG7239 only conserved amongst Drosophilidae;

EP(2)2219 2L (33E4) CG6405 overexpression, inserted 1.5kb upstream of CG6405

weak variable n.d., two mammalian orthologues

EP(2)2190 2R (55E1) CG30332 overexpression, 1kb 5’ of CG30332

strong hooks n.d.

EP(2)2182 2R (54A2) CR30234 (cytosolic tRNA gene)

overexpression very strong variable n.d.

EP(3)313 3R (98E5) CG1523 (related to WD40 repeat-containing protein 32)

overexpression, inserted directly 5’ of gene

strong variable n.d.

45

EP(2)2269 2R (53D11)

CG34460 antisense to CG34460?, EP is ~2.5kb away

weak variable n.d.

EP(2)383 2L (23C4) nothing downstream for >10kb, next CG is CG17265 14kb away

inserted 3’ of CG3558

weak short, expanded at turn

n.d.

EP(2)2173 2L (35B2) nothing downstream for >10kb

inserted into 5’ end of no ocelli, wrong strand

weak variable n.d.

EP(2)2146a ?? ?? genome position of EP unclear

strong ‘searching cells’ n.d.

EP(2)2265 ?? ?? genome position of EP unclear

very strong short, straight, expanded

n.d.

EP(2)985 ?? ?? genome position of EP unclear

strong budding, branching

n.d.

* Penetrance of phenotypes: weak = 20-30% of embryos showing phenotype

strong = 30-50% of embryos showing phenotype

very strong = >50% of embryos showing phenotype (with 3 cases < 70% and 4 cases >90%)

46

FIGURE LEGENDS

Figure 1. Salivary gland development visualized using fkhGal4-driven GFP-

marker expression.

Salivary gland morphogenesis from embryonic stage10-15 is shown. A Schematic

of salivary gland invagination, ventral view. B-F shows low magnification confocal

sections of embryos stained with phalloidin to reveal actin (red) and expressing

GFP-EFGas2 under the control of fkhGal4 in the salivary glands (green). C-E show

lateral views, B is a ventral and F a dorsal view. G-K’ Close up confocal sections of

salivary glands labelled with phalloidin to reveal actin (red) and expressing SrcGFP

under the control of fkhGal4 (green, and as a single channel in G’-K’)). All panels

show lateral views. Note that GFP-EFGas2 labels microtubules, whereas SrcGFP is

targeted to the membrane and thus reveals cell shape.

Figure 2. Phenotypes observed upon overexpression of genes in the salivary

glands using fkhGal4.

A Schematic of the set-up of the screen. The phenotypes observed in the screen

could be classified according to the categories depicted in this figure. Broad

categories are ‘invagination defects’ (B, C), ‘gland shape & lumen defects’ (D-I) ,

‘positioning defects’ (K-P) and ‘gland fate defects’ (Q). All panels show the GFP-

marker expression in green and phalloidin staining to reveal actin in red. Lateral or

dorsal views are indicated in each panel. The line in C indicates the area of the too

wide opening of the invaginating gland shown; the arrow in D points to where

proximal and distal cells of the gland touch due to excessive bending; the double

47

arrows in H indicate the too wide width of the gland shown; the arrows in M point to

two buds emerging from the side of the gland shown; the arrows in N point two the

two ends of a fork; the arrows in O point to cell of the glands that appear to touch

across the midline. B-G and K-Q are widefield fluorescence images, H, I and Q are

confocal sections. Embryonic stages of embryos shown are indicated in the panels.

Figure 3. Chickadee (Profilin) is important for salivary gland invagination.

chickadee encodes the Drosophila Profilin protein. A Scheme of the chic locus

indicating the position and orientation of the two EP lines that showed phenotypes

when driven in the salivary glands. B, C and D, E show phenotypes observed in the

screen for EP713 and EP1011 respectively, widefield fluorescent images of live

embryos are shown. F-G’ overexpression of chickadee using a UAS-chickadee

construct led to invagination problems and aberrantly shaped glands. F show an

internal confocal stack of a gland (14µm thick), labelled with Crumbs to reveal the

apical surface/lumen of the glands (red) and showing the SrcGFP marker in green.

G, G’ show a surface stack of the same embryo (with Crumbs in red and SrcGFP in

green in G and crumbs as a single channel in G’; 3µm thick). The arrow points to

disrupted epidermis in the region of the placode from where the glands have started

to invaginate. Note the absence of Crumbs from the apical surface of cells in this

region. H, H’ shows a low and high magnification view of the slightly disorganized

epidermis of a chic mutant embryo labelled for crumbs, with H’ showing the salivary

gland placode and gland in a projection (18µm thick stack). I shows a wild-type

placode and gland (17µm thick stack) at the same stage as in H’. Note that the

highly organized arrangement of apical constriction of the placodal cells is less

48

apparent in the chic mutant (bracket in H’ and I). K-L and O-P show the aberrant

glands and disrupted epidermis in two different chic alleles (chic01320 and chic221) at

stage 12, labelling for Crumbs is in green (and also shown as a single channel in K’

and O’) and for phalloidin is red in K, L, O, P. Note the disruption and absence of

apical Crumbs labelling in the region of the salivary gland placode (arrows in K’, L,

O’ and P point to these areas). K is a projection of a 35µm thick stack, L is a 5µm

thick surface projection, O is a projection of a 26µm thick stack, and P is a 3µm thick

surface projection. M-N show the disrupted epidermis in the region from where

salivary gland cells invaginated in a stage 14 embryo (Crumbs is in green in M and

as a single channel in M’, and phalloidin is in red in M and as a single channel in N;

M is a projection of a 34µm thick stack, N is a 5µm thick surface stack). For

comparison a stage 14 wild-type embryo is shown in Q-Q’’ (Crumbs is in green in Q

and as a single channel in Q’, and phalloidin is in red in Q and as a single channel

in Q’’; Q is a 5µm thick surface stack). The arrows in M’ and N point to the disrupted

region, the white lines in M and Q indicate the ventral midline (the view in M-N is

slightly oblique). R-V’’ show chic221 mutant embryos at stage 12-14. R, R’ are lateral

views of a placode, whereas S shows an internal stack of the gland. T, T’ are ventral

views of the two placodes, with U showing an internal stack of the glands. V-V’’

show a surface stack of a mutant embryo (5µm thick). Crumbs is in green in R, T

and V and as a single channel in R’,T’ and V’, phalloidin is in red; both S and U

show Crumbs labelling to outline the lumen of the gland. DE-Cadherin (DE-Cad)

labelling is in red in V and as a single channel in V’’.

49

Figure 4. rhomboid1 overexpression disrupts salivary gland morphogenesis,

but is not sufficient to induce salivary duct fate.

A Scheme of the rho locus indicating the position of the EP identified in the screen.

B Scheme depicting the known involvement of EGF signalling in salivary gland

morphogenesis. EGF is released from the midline (red line) and induces, in the cells

close to the midline (light green), the repression of fkh which in turn leads to

suppression of secretory fate in these cells, inducing them to adopt duct fate. Fkh

expression remains high in the remaining salivary gland primordium (dark green),

thus inducing these cells to form the secretory part of the gland (KUO et al. 1996). C-

C’’ Overexpression of rhomboid1 in the salivary glands using EP(3)3704 led to

glands that, at stage 15 of embryogenesis, were located too far anterior with

secretory cells that appeared cuboidal instead of columnar, no proper duct

connecting the secretory portions to the outside and a aberrantly shaped lumen.

The SrcGFP marker is in green in C and as a single channel in C’, phalloidin is in

red in C and as a single channel in C’’. D-D’’ The same phenotype as in C is

observed when a UAS-rhomboid1 construct is expressed in the glands using

fkhGal4. The GFP-EFGas2 marker is in green in D and as a single channel in D’,

Crumbs is in red in D and as a single channel in D’’. E-F’ Already at stage 13 the

invaginated portion of the gland shows aberrant morphology (‘ectopic’ lumen

indicated by the arrow in E’), and the amount of cells remaining at the surface

appears too large (bracket in E’ and F’). The SrcGFP marker is in green in E and F

(and as a single channel in E’ and F’), crumbs is in red. G-P’’ analysis of dCreb-A

and Eyegone expression: markers of secretory and duct fate, respectively. G-I’’

Control glands expressing only the GFP-EFGas2 marker labelled with antibodies

50

against dCreb-A and Eyg at stage 11 (G-G’’), stage 14 (H-H’’) and stage 15 (I-I’’).

K-M’’ Glands expressing UAS-rhomboid1 in the salivary glands using fkhGal4

labelled for dCreb-A and Eyg at stage 11 (K-K’’), stage 14 (L-L’’) and stage 15 (M-

M’’). Note that despite the irregular shape and ectopic cells (bracket in L) dCreb-A is

strongly expressed in the early invaginated part of the glands (L’ and M’). Eyg is

expressed in the most anterior cells of the invaginated glands (L’’ and M’’), as in the

control, and also in the ectopic cell ‘bulge’ on the surface of the embryo (L’’, bracket

in L denotes the ‘bulge’, the dotted line indicates the ventral midline). N-P’’ Glands

expressing UAS-rhomboid1 using armGal4. N-N’’ dCreb-A and Eyg expression in

the placode at stage 11. O, O’ Ventral view of the remaining placode (O) and

invaginated glands (O’) at stage 13. More ectopic cells expressing Eyg are found on

the ventral surface (bracket in O). Small stubby glands have invaginated and

express dCreb-A (arrows in O’). P-P’’ Lateral view of glands at stage 14. More cells

have invaginated and express dCreb-A (arrow in P), and the most ventral cells on

the surface still express Eyg (P’’). GFP makers are in green, dCreb-A is in red and

Eyg in blue in G-P, dCreb-A is shown as a single channel in G’-N’ and P’, and Eyg

as a single channel in G’’-N’’ and P’’. All panels are projections of confocal stacks

that cover the whole thickness of either the invaginated glands or of the placode at

earlier stages.

Figure 5. EGFR signalling is necessary but not sufficient to induce salivary

duct fate, and overactivation leads to ectopic cell divisions.

Analysis of components of the EGFR signalling pathway in the glands. It has been

reported previously that salivary glands in rhomboid/spitz-group mutant embryos do

51

not specify any ductal portion of the glands (KUO et al. 1996). A-B’ This phenotype

was confirmed in a rhoPΔ5 embryos, a null allele of rho (FREEMAN et al. 1992). A, A’

At stage 13 most of the secretory cells of the gland have invaginated, leaving two

large holes visible at the surface of the embryo (arrows in A; A shows the surface of

the embryo, A’ shows an internal confocal stack to reveal the shape and location of

the invaginated glands). B, B’ At stage 14 the glands have fully invaginated and

detached from the surface of the embryo, leaving no ductal connection to the

outside and a large hole on the surface (B shows the surface of the embryo, B’

shows an internal confocal stack to show the blunt ended gland; arrow in B’ points

to the blunt end, arrow in B points to the hole). A-B’ show labelling for Crumbs. C

shows an amended gland fate specification scheme as introduced in Fig. 4 to

illustrate the altered signalling in rho mutant embryos, where absence of EGFR

signalling induces the entire salivary gland primordium to adopt secretory fate. D-F

Overexpression of argos, an extracellular inhibitor of EGFR signalling (SCHWEITZER

et al. 1995), using fkhGal4 caused a similar phenotype as that seen in rho mutants:

D-D’’ At stage 13 the invaginated secretory portion of the glands detaches from the

surface of the embryo (arrow in D’), and no duct is formed. E, E’ The salivary gland

primordium at stage 11 appears normal. F Scheme showing that downregulation of

EGFR signalling leads to conversion of presumptive duct cells into secretory cells.

G-I Overexpression of an activated form of the EGF receptor (CA-EGFR) using

fkhGal4 leads to glands with highly disorganized and aberrant lumen from stage 12

on. G-G’’ shows a confocal stack of stage 13 embryo, the arrow in G points to the

lumen marked by Crumbs. H, H’ Cell shapes marked by crumbs in the salivary

gland primordium at stage 11 appear normal. I Scheme showing that elevated

52

EGFR signalling throughout the primordium does not induce duct fate in all cells. K-I

Overexpression of a secreted and active form of the ligand Spitz using fkhGal4

leads to glands that very much resemble those seen upon overexpression of rho

(compare Fig. 4). K-K’’ shows a confocal stack of stage 15 embryo. No ductal

structures are formed, and the shape of the secretory cells and the lumen is

aberrant. L, L’. The salivary gland primordium at stage 11 appears disrupted with

irregular and too large apices of the invaginating cells. The arrow in L points to a

group of cells that show midbodies left by mitotic divisions marked by the GFP-

EFGas2 microtubule marker. Ectopic mitoses can also be observed when CA-EGFR

is expressed using fkhGal4 . M Scheme showing that elevated EGFR signalling

through overexpression of secreted Spitz throughout the primordium does not

induce duct fate in all cells. The SrcGFP or GFP-EFGas2 markers are green in D, E,

G, H, K, L, and shown as a single channel in D’, G’, K’, Crumbs labelling is red in D,

E, G, H, K, L and shown as a single channel in D’’, E’, G’’, H’, K’’, L’. M-O’’’

Analysis of ectopic cell divisions induced by activation of EGF signalling using

phospho-histone H3 (p-HisH3) as a marker of mitosis. In the marker-expressing

control p-His3 labelling is restricted to the area outside the placode (M) and

invaginated gland (P). When UAS-secreted spitz (N, Q) or UAS-rhomboid1 (O, R)

are expressed in the glands, many mitotic cells can be found in the placode and

invaginated gland. GFP-markers are in green, p-HisH3 in red in M-R and O’,

Crumbs in blue in M-Q and R, and DAPI in blue in O and O’. O’-O’’’’ show a higher

magnification of the dividing cells in O. GFP-EFGas2 is shown as a single channel

in O’’, p-HisH3 as a single channel in O’’’ and DAPI as a single channel in O’’’’. The

53

dotted line in O’-O’’’’ highlights a cell in anaphase (note the spindle in O’’), the

arrow in O’-O’’’’ points to a midbody in telophase, similar to the ones indicated in L.

Figure 6. egalitarian overexpression reveals a potential role for egalitarian

and BicD in salivary gland morphogenesis.

A Scheme of the egalitarian (egl) locus indicating the gene structure and the

position of the EP identified in the screen. B, C Two phenotypes observed in the

screen upon overexpression of egl using EP(2)938: bent (B) and shortened (C)

glands. The GFP-EFGas2 maker is in green, phalloidin labelling in red. B and C are

widefield fluorescent images. D, D Overexpression of a UAS-egl construct using

fkhGal4 frequently led to short glands at stage 14, with some GFP-positive cells

losing contact with the glands (arrow in D). E, E’ shows a comparable wild-type

embryo. Crumbs is in red in D and E and as a single channel in D’ and E’, SrcGFP

is in green in D, and Shot is in green in E. F-G’ Dorso-lateral views of stage 14

embryos. F, F’ egl mutant embryos often show a disrupted epidermis with

mislocalized Crumbs labelling (arrows in F’ point to areas where Crumbs is

completely absent), whereas in wild-type embryos Crumbs is localized apical-

circumferential in all epithelial cell (G, G’). H, H’ egl mutant embryo showing a

disorganized salivary gland placode, with a too large and extended invagination

hole. I, I’ A stage 13 egl mutant embryo with a gland that appears to wide and short,

showing mislocalized Crumbs labelling. K, K’ Ventral view of a stage 13 egl mutant

embryo. K is a surface confocal stack, showing two disrupted areas in the epidermis

where the glands invaginated (arrows). K’ shows an internal confocal stack of the

same embryo, with a too wide and aberrant gland (the red dotted line traces the

54

outline of the gland). L, L’’ Higher magnification of the gland shown in K’ in a

smaller confocal stack. Note the mislocalized Crumbs protein at the lateral sides of

cells and internally (arrow in L’) that cannot be seen in wild-type glands (compare to

M’). H-L’’ Crumbs labelling is in red in H, I, K, L, and as a single channel in H’, I’,

K’, L’, phalloidin labelling is in green in H, I, K, L, and as a single channel in L’’. M,

M’ Magnification of a section through a stage 13 wild-type gland, SrcGFP is in green

in M, Crumbs is in red in M and as a single channel in M’.

Figure 7. bitesize overexpression reveals a potential role for bitesize in

salivary gland morphogenesis.

A Scheme of the bitesize (btsz) locus indicating the gene structure and the position

and orientation of the EP identified in the screen. B-C’ Potential knock-down of

Bitesize through overexpression of antisense RNA using EP(3)3567 lead to

epithelial defects in the overexpressing cells (arrow in B’) and glands that invaginate

with aberrant morphology. B, B’ shows a surface confocal stack of a stage 13

embryo, C, C’ shows the corresponding internal stack to reveal the glands. Note the

absence of Crumbs labelling in the area that shows GFP-EFGas2 marker

expression (arrow in B’). GFP-EFGas2 is in green in B and C, Crumbs is in red in B

and C and as a single channel in B’ and C’. D-E’ show ventral views of btszK13-4

mutant versus wild-type embryos at stage13. The arrow in D point to the disrupted

epidermis in the mutant embryo. Note the disorganization of the placode area

compared to wild-type (indicated by the brackets in D ad E), and the failure to

accumulate Shot apically (arrow in E’’ indicates the accumulation in the wild-type).

Crumbs is in red in D and E, and as a single channel in D’ and E’, Shot is in green

55

in D and E and as a single channel in D’’ and E’’. F-I’’ show examples of lateral

views of btszK13-4 mutant versus wild-type embryos at stage 14. F-F’’ Highly

disrupted and disorganized epidermis in the btszK13-4 mutant (arrows point to areas

lacking apical circumferential Crumbs labelling, compare to the wild-type epidermis

in I-I’’). G-G’’ show an internal stack of the same embryo as in F (the corresponding

internal stack for the wild-type embryo in I is shown in K-K’’). Note that the salivary

gland of btszK13-4 mutant embryo is losing apical Crumbs accumulation (G’)

compared to the wild-type (K’), the phalloidin labelling in G’’ still shows cell outlines,

but these also lack apical actin accumulation as seen in the wild-type (K’’). Crumbs

labelling is in green in F, G, I, K, and as a single channel in F’, G’, I’, K’, phalloidin is

in red in F, G, I, K and as a single channel in F’’, G’’, I’’, K’’. H and L show confocal

stacks of the embryos in F and I at the level where the salivary duct reaches the

epidermis labelled for Crumbs, H shows the of btszK13-4 mutant and L the wild-type.

Note that the duct shown in H lost apical Crumbs accumulation (the arrow points to

the remnants of Crumbs labelling in the duct) and that the epidermis at the point

from where the glands invaginated is disrupted and lacks apical Crumbs (indicated

by the bar in H). M-N’’ The btszJ5-2 mutant at stage 14 also shows disrupted

epidermis and loss of Crumbs (M’’) and also DE-Cadherin (M’) in the area where

the placode was previously located. Crumbs labelling in the invaginated gland is

aberrant (N’’) whereas DE-Cadherin appears still apical (N’). M-M’’ is a projection of

a 5µm thick confocal surface stack, whereas N-N’’ shows the projection of a 20µm

thick internal stack covering the whole gland. DE-Cadherin is in green in M and N

and as a single channel in M’ and N’, Crumbs is in red in M and N and as a single

channel in M’’ and N’’.

56

Figure 8. capricious overexpression reveals a potential role for capricious

and tartan in salivary gland morphogenesis.

A Scheme of the capricious (caps) locus indicating the gene structure and the

position of the EP identified in the screen. B, C show live images of the GFP-

EFGas2 marker of the caps overexpression phenotype using EP(3)552 observed in

stage 15 embryos in the screen. D-E’ Confocal stacks of two examples of aberrantly

shaped lumen of salivary glands at stage 15 upon overexpression of a UAS-caps

construct using fkhGal4. The lumen is highlighted by crumbs labelling in D’ and E’

and very much resembles the defects observed in the screen. F-H’ Shown are

examples of invaginating glands at stage 12. F, F’ is a surface stack of the

primordium upon UAS-caps overexpression. Note that the hole at the invagination

point is too extended and not positioned completely within the primordium (as

highlighted by the GFP marker) compared to the wild-type primordium shown in H,

H’, G, G’ show a complete stack of the glands at stage 12 upon UAS-caps

overexpression. Note that the size of the invagination hole (marked by the red

dotted lines in G’) is again too large and irregular compared to wild-type, and the

invaginated portion of the glands shows a too wide and irregular lumen. The GFP-

EFGas2 marker is green and Crumbs labelling in red in D, E, F, G, H and Crumbs is

shown as a single channel in D’, E’, F’, G’. I-M capsPB1 single, trn28.4 single and

capsDel1 trn28.4 double mutants (all are null mutations, (MAO et al. 2008)) often show

defects in salivary gland morphology, i.e. irregular lumen at different stages of

invagination. Staining for the fly spectraplakin Shot is in green in and Crumbs in red

in I, K, L and M, and Crumbs is shown as a single channel in I’ and K’. N-S A lacZ

57

containing P-element insertion into the caps locus reveals that caps is not

expressed in most cells of the salivary glands. N and M show β-galactosidase (β-

gal) labelling at stage 12 and 15, respectively. P-S show cross sections of a gland at

stage 14. Note that the glands are surrounded by cells expressing caps. N-S The

outline of the glands is marked by a white dotted line, β-gal labelling is in green and

Crumbs in red. T-U A lacZ containing P-element insertion in the trn locus reveals

that trn is expressed in salivary gland cells at all stages. T, T’ Most cells of the

salivary gland placode at stage 11 express trn at varying levels (border of the

placode is marked by dotted lines). U At stage 14 trn is still expressed strongly in all

salivary gland cells including the duct. The outline of the gland is indicated by a

dotted line. β-gal labelling is in green and Crumbs in red in T and U, β-gal is shown

as a single channel in T’.

Supplementary Figure 1. In situ hybridization for chic mRNA in control and

chic-overexpressing embryos.

A-B’’ chic mRNA expression pattern in wild-type ‘marker-line’ embryos at stage 14

(A-A’’) and stage 15 (B-B’’). A and B show overlays of the in situ signal (that is

shown as a separate channel in A’ and B’) false-coloured in red with the SrcGFP

marker signal in green (also shown as a single channel in A’’and B’’). C-E show

chic mRNA levels upon chic overexpression using EP(2)713 x srcGFP fkhGl4 at

stage 12 (C), stage 14 (D) and stage 15 (E).

Supplementary Figure 2. In situ hybridization for btsz mRNA in control and

E(3)3567-overexpressing embryos.

58

btsz mRNA expression pattern in control (A and D; srcGFP fkhGl4) and EP(3)3567

x fkhGal4 (B and C; E) embryos at stage 13 (A-C) and stage 15 (D and E). Note that

btsz mRNA is strongly enriched at the apical surface of the salivary gland cells in

the control (arrows in A and D) and appears reduced in the glands in the

EP(3)3567-expressing embryos (arrows in B, C and E).

Supplementary Figure 3. In situ hybridization for caps mRNA.

caps mRNA expression pattern in wild-type ‘marker-line’ embryos at stage 14 (A-

A’’) and stage 15 in a ventral (B-B’’) and lateral (C-C’’) view. A, B and C show

overlays of the in situ signal (that is shown as a separate channel in A’, B’ and C’)

false-coloured in red with the SrcGFP marker signal in green (also shown as a

single channel in A’’, B’’’and C’’).

Supplementary Figure 4. In situ hybridization for trn mRNA.

trn mRNA expression pattern in wild-type ‘marker-line’ embryos at stage 13 (A-A’’)

and stage 15 (B-B’’). A and B show overlays of the in situ signal (that is shown as a

separate channel in A’ and B’) false-coloured in red with the SrcGFP marker signal

in green (also shown as a single channel in A’’and B’’).

Supplementary Table 1. Table of all EP lines analysed in the screen.

This table list all EP lines that were analyzed in the screen. Lines without a

phenotype are marked in blue as ‘generally unremarkable’, genes picked up in the

first pass of live screening that were not confirmed in the second examination are

marked in light yellow as ‘HIT first pass, not confirmed’, and the confirmed genes

59

are marked in bright yellow as ‘HIT’. Green labelling of a gene indicates that several

P-insertion into this locus were analyzed in the screen. The ‘existing data’ column

lists which gene the EP is annotated to be inserted in according to Flybase

(http://flybase.bio.indiana.edu/), but as this does often not represent the gene that

would be overexpressed, we have added the expression information where

possible.

60

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