A Novel Role For Drosophiia Akt During Embryonic Development:

Positive Regulaiion of the Trh Transcription Factor

Ben K. Wetsch

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Zoology

University of Toronto

O Copyright by Ben K. Wetsch, ZOO1

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Abstract

"A Novel Role For Drosophila .Ikr During Embryonic Development: Positive Regulation of the

Trh Transcription Factor:'

Ben K. Wetsch

blaster of Science

Department of Zoology. University of Toronto. 300 1

Since 199 1. much work has focused on the signaling pathway for the activation and

function of Protein Kinase B (PKB)/.Akt. the mammalian homologue of a retroviral oncogene. v-

ukr. Despite intensive research on the function(s) of this enzyme. few in vivo phosphorylation

targets have been identified. The recent identification of a mutation in the Drosophiln

rnelanogmrer homoiogur of PKB. DaktlY. has provided a new genetic tool for ideiiti@ing gene

products that have an in vivo role in the DAktl signaling pathway. A grnetic screen has

implicated the rrcicheuless ( trh) gene. which is essential for tubulogenesis dunng development of

the Drosophila tracheal systern. as being a component of this signaling pathway. Analysis of

data From both genetic and ectopic expression studirs revealed that D A M is a positive regulator

of Trh transcriptional activity and that the mechanism by which D A M regulates this activity is

through the conuol of Trh nuclear translocation.

Acknowledgements

1 would like to thank my committee members: Dr. h e n Manoukian. Dr. Ellen Larsen

and Dr. Sue Varmuza. 1 would especially like to thank my supervisor Dr. Amen Manoukian for

his guidance and advice on this research project over the past three years and my CO-supervisor

Dr. Ellen Larsen for her advice and comments during the writing process. Suggestions and

criticisms by Dr. Rich Binari during the writing process and research-related discussions over the

past three years have bren helpful. indispensable and grearly appreciated. 1 would also like to

thank Norman Anthopoulos for his help with microinjections. confocal microscopy and

computrr-relatrd problems. My main collaborator on this project. Jing Jin from Dr. Jim

Woodgett's lab. has worked very hard on the biochemistry portion of this project and was

rlrtremely helpful whenever I needed anything. In addition. Dr. Daniel Isaac from Dr. Debbie

Andrews' lab was extremely helpful in supplying Trh-related advice and reagents including

Drosophila stocks. antibodies and cDNA's. I thank Paul Guy LaPointe for his help with the

Western blot and for his man. intellectual discussions andor debates about science. politics and

life. Last. but not least. 1 would like to thank my parents. my brothers (Jeff and Justin) and

Regan for their support. patience and guidance in the times when 1 needed it most.

Table of Contents:

Section 1 : Introduction (Pages 1 - 17)

Section 7: Materials and Methods (Pages 18-33)

Section 3: Results (Pages 34-5 1)

Section 4: Discussion (Pages 52-68)

Section 5 : Bibliography (Pages 68-87)

List of Tables:

Table 1-Downstream Targets of PKB (Introduction) Pg. 6

Table 2-Fly Stocks Used in This Study (Materials and Methods) Pg. 20

Table Konfidence Interval Comparisons (Materials and kkthods) Pg. 13

Table 4-Primary Antibodics Used in This Study (Materials and klethods) Pg. 24

Table 5-SDS Gel Electrophoresis (Materials and Methods) Pg. 32

Table 6-P elçmentlDaktZq Enhancer of Lethality Screen (Results) Pg. 37

List of Figures:

Figure 1 -Structure of PKB and DAkt 1 Proteins (Introduction) Pg. 2

Figure 2-PKB Signaling Pathway (Introduction) Pg. 3-4

Figure 3-Trachral Development (Introduction) Pg. 1 1

Figure 4-Trh Structure (Introduction) Pg. 13

Figure 5-Trh Signaling Pathway (Introduction) Pg. II

Figure 6-FGF-Dependent Tncheal Branch Migration (Introduction) Pg. 1 7

Figure 7-Dakf lq Tans-Heterozygous Enhancer Screen ( Results) Pg . 3 5

Figure 8-Genetic Analysis of Trh Activity (Results) Pg. 39-40

Figure 9-Transgenic Expression of ~ r h ~ " " and Trh Ser665AIa (Results) Pg. 4647

Figure 10-Anti-FLAG h t ibody Staining (Results) Pg. 50-5 1

Figure 1 I-RTK Signaling During Tracheal Development (Discussion) Pg. 57

Figure 12-Mechansim For Regulating Trh Nuclear Translocation (Discussion) Pg. 63

1. Introduction

Protein kinase B (PKB)/c-.4kr is the human cellular homologue of a retroviral oncogene.

v-dkt, which was isolated from the acute transforming retrovirus AKTI (Bellacosa et al.. 199 1 :

Staal. 1987). It was simultaneously identified because of its srquence similarity to the related

kinases PKA and PKC (Coffer and Woodgett. 199 1 : Jones et al.. 199 1 ). Three mammalian PKB

isoforms. encoded by three different grnes (termed c ~ d K T 1 . PI.4KTZ and y ) . have been

identified: each isoform is almost ubiquitously expressed such that al1 tissues express at least one

form of PKB (Altomare et al.. 1995: Bellacosa et al.. 1993: Bellacosa et al.. 199 1: Coffer and

Woodgett. 199 1 : Konishi et al.. 1995). With the exception of the y isoform. PKB is 180 arnino

acids long and has four main conserved domains: the N-terminal PH domain (amino acids 1-

106). a glycine-rich domain (residues 107- 1-17}. the central catalytic serine/threonine kinase

domain (amino acids 148-4 1 1) and the "regulatory" domain at the C-terminus (residues -Il2-180)

(Figure 1) (Bellacosa et al.. 1993: Bellacosa et al.. 199 1 : Coffer and Woodgett. i 99 1 : Haslam et

al.. 1993: Jones et al.. 1991: Mayer et al.. 1993). Besidrs having homology to the r-.4kr

oncogene. further evidence for the oncogenic potential of PKB is that it is over-expressed in

some mamrnalian pancreatic. ovarian. breast and gastric cancers (Bellacosa et al.. 1995: Cheng et

al., 1992: Miwa et al.. 1996: Staal. 1987).

PKB is nomally inactive in mammalian cells. I t must be activated and its activation is

dependent on the binding of various extracellular growth factors and cytokines to their respective

membrane-bound receptors (Figure 2) (Ahmed et al.. 1997: Alessi et al.. 1996: Andjelkovic et

al.. 1997: Burgerinç and Coffer. 1995: Cross et al.. 1995: Franke et al.. 1995: Kohn et al.. 1993:

Kohn et al.. 1996: Songyanz et al.. 1997). Upon activation. the receptor dimerizes.

autophosphorylates specific intracellular tyrosine residues and subsequently recmits Src

Figure 1-Diagramm atic representation of PKB and DA kt 1 protein structure

rhrj08

1 ~ h 9 - î '

Figure: The general size and domain structure of the m a m d i a n PKB isoforms and the Drusuphilu hom O logue. The N-terminal Plecks trin

4 1 1 PKB

I I

Homology (PH) domai n. the central cataly tic kinase domain and the C- temi na1 activating phosphoryl ation sites (TIdo8KhP2 and Ser4'4SePo5) are indicated.

I 1 DAkt 1

530 1 1 ami no PH Cataly tic Ki nase Domai n aci ds

I

Figure 2-Cunent mode1 for PKB signal h g

~ G r o w t h Factor

Extrace

T TEN

Piasm a Membrane

Figure: Figure2A shows the inactive state of the PI-3WPKB signaling pathway pnor to

growth factorkytokine activation. PKB remains cytosolic and inactive until receptor t

activation and PI-) K recruitment to the membrane. P I 4 K phosphory lates PIP2

(phospliatidylinositol 4.5-biphosphate) to grnerate PIP3 (phosphatidylinositol 3.4.5-

triphosphate). in the absence of PTEN inhibition (Figure-B). PIP3 molecules are thought

to recruit PKB and its activating phospholipid dependeni kinases: PDKl and PDK2.

Following phosphorylation and activation. PKB is free to seek out substrates at the

plasma membrane. in the cytosol or in the nucleus (FigureX).

homology 2 (SH2) domain-containing proteins such as the p85 regulatory subunit of

phosphatidy linositol 3-kinase ( P I - X ) (Pawson and Scott. 1997). The p 1 10 catalytic subunit of

PI-3K phosphorylates the membrane lipid phosphatidylinositol4.j-biphosphate (PIP2) at the D-3

(3') position of the inositol ring to produce the second messenger PI (3.45)-triphosphate (PIP3)

in the plasma membrane (Auger et al.. 1989: Carpenter and Cantley. 1990; Whitman et al..

1988). The PH domain of PKB can bind the phospholipid products of P I - X and most likely

targets PKB to the membrane following Pi-3K activation (Franke et al.. 1997: Klippel et al..

1997). Recently. the tumor suppressor PTEN. a 3' phospholipid phosphatase. has been shown to

target the PI (3.4.5) P3 products of PI-3K and thus act as an intracrllular inhibitor of the PI-

3 WPKB signaling pathway (Figure 2) (Stambolic et al.. 1998).

PKB localiwtion to the plasma membrane is required for its full activation following its

phosphorylation at arnino acid residues threonine 308 and serine 473 (Figure 2) (Alessi et al..

1997: Andjelkovic et al.. 1997: Kohn et al.. 1996: Stephrns et al.. 1998). Phosphorylation at

Thr308 is mediated by a PI (3.4.5) P3-dependent kinase (termed PIP3-dependent kinaseiPDK1)

that also contains a C-terminal PH domain (Alessi et al.. 1997: Stephens et al.. 1998). The CO-

attraction of PKB and PDK 1 to the PI-3 K-genrrated phospholipids may be a way of bringing

these kinases into proximity of one another. A kinase that phosphorylates SerJ73 in vivo

(tentatively named PDK?) has not yet been identified: however. MAPKAP kinase-? and ILK

have been shown to phosphorylate PKB at Ser473 in vitro (Alessi et al.. 1996: Delcommeme et

al.. 1998). Following its activation at the plasma membrane. PKB can intrract with cytosolic

phosphorylation targets and nuclear ones. following its subsequent translocation to the nucleus

(Figure 2)(Andjelkovic et al.. 1997: Meier et al.. 1997).

PKB has multiple functions in the cell. from the regulation of metabolic pathways

controlling glycogenesis and protein synthesis. to a key role in ce11 survival, proliferation and.

more recently, a role in ce11 size regulation (Brunet et al.. 1999: Cardone et al.. 1998; Cross et al..

1995; Datta et al.. 1997: Deprez et al.. 1997: Kang et al.. 1999: Kohn et al.. 1996: Kops et al..

1999: Romashkova and Makarov. 1999: Scott et al.. 1998; Shioi et al.. 2000). The specific

mechanism by which PKB mediates its various cellular responses has been studied by

identifying direct or indirect downstream targets of the PI-? WPKB signaling pathway. The

minimum target sequence motif for PKB phosphorylation has been identified as Arg-X-Arg-Y-

2-Ser/Thr-Hyd. where X is any amino acid. Y and Z are small residues other than glycine. and

Hyd is a bulky. hydrophobic residue (Alessi et al.. 1996). To date. a number of direct and

indirect downstream targets have been identified: each target. dong with its specific function. is

listed in Table 1. However. the only protein that is genrraliy acceptrd as a truè in vivo

phosphorylation target is the FKHRL 1 transcription factor and it is interesting to note that this

interaction was initially implicated by a gr netic interaction between mutations in the genes

encoding the Cuenorhabdiris rlegc~ns homologues of PKB ( A U - I and AKT-2) and FKHRLI

(DAF- 16) (Paradis and Ruvkun. 1998).

Table 1: Downstrem targets of PKB

Downstream Targets

GSK-3

GLUTLF

BAD

PFK-2

Caspase9

F M P

FKHRL 1

AFX

Function

Gl ycogen synthase regulator

Glucose transporter

Pro-apoptotic mitochondrial protein

Phosphofnictokinase- 1 regulator

Apo ptotic protease acrivator

Translation initiation regulator

Pro-apoptotic transcription factor

Transcription factor

Reference

(Cross et al.. 1995)

(Kohn et al.. 1996)

(Datta et al.. 1997)

(Deprez et al.. 1997)

(Cardone et al.. 1998)

(Scott et al.. 1998)

(Brunet et al., 1999)

(Kops et al.. 1999)

6

hTERT 1 Human telornerase reverse transcriptase 1 IKK 1 Apoptotic inhibitor

(Kang et al., 1999)

(Romashkova and

Makarov, 1999)

(Dimmeler et al..

1999)

(Fulton et al., 1999)

The Dakrl gene iç the Drosophila melcinogosrrr homologue of PKB/;lkr. It spans

approximately 5.6Kb of genomic DNA and maps to cytological position 898 on the right arm of

the third chromosome. Like its mammalian homologue. DAktl contains an N-terminal PH

domain. a central catalytic serine/threonine kinase domain and two activation sites at Thr 342

and Ser 505 (Figure 1). DAkt 1 has 75% homology to mammalian PKB at the amino acid Ieve!

and 64% identity at the DN.4 level. Specifically. the N-terminal PH domain is 7 1% homologous

and the catalytic domain shows 86% homology with conserved motifs for ATP binding and for

defining the SeriThr specificity of the kinase. respectively. Dukfl is ubiquitously-expressed.

suggesting that localized activation ma. be a regulatory mechanism. Both its transcription and

the intrinsic kinase activity are differentially-regulated throughout embryogenesis. larval

development. pupation and between the sexes in adults. These transcxipt and protein expression

studies. as well as in sifzr localization of h k f l transcripts. suggrst that Daktl is regulated both

matemally and zygotically (Franke et al.. 1994).

A Drosophila mutation of Dakf 1 . Dakt I V . was identified as an EMS-generated recessive

larval-lethal mutation that failed to complement a large deletion at 89B and which can be rescued

by a Daktl or mamrnalian PKB transgene. This mutation is the result of a Phe-Ile substitution at

position 327 inside the 'DFG' motif of the cataiytic domain that results in a significant loss of

D A M kinase activity (Staveley et al., 1998). Germline clone (GLC) analysis (Chou and

7

Perrimon, 1996) was used to eliminate both the materna1 and the zygotic contribution of DAkctl

and subsequently show that this ubiquitously-expressed cell survival factor is required for

preventing ectopic apoptosis during embryogenesis (Stavelry et al.. 1998).

Programrned ce11 death. or apoptosis. is a normal evolutionarily-conscrved process that

plays a critical role during metazoan development. Apoptosis can occur as a regular and

reproducible part of the developmrntal program or it can be induced as a suicide protection

factor in the face of homeostatic disruption. The exrcution of the apoptotic prognm leads to the

deletion of unneeded. damaged. infected or abnormal cells. Apoptosis is morpho1ogicalIy

distinct from another type of ce11 death. ce11 necrosis. which is induced by extemal injury and

which is chancterized by the swelling and rupture of the ce11 and its organelles. Conversely.

programmed ce11 death is characterized by the condensation. fragmentation and eventual

phagocytosis of the apoptotic crlls and thrir fragments. while maintaining plasma membrane and

organelle integrity (until very late stages of apoptosis) (Jacobson et al.. 1997: Kerr et al.. 1972:

Steller. 19%). During Drosophilcr development. apoptosis begins at stage 1 1 (7 hours AEL) and

continues throughout embyogenesis in an overall reproducible pattern that affects many

different tissues including the central nervous system. developing head tissue and the epidermis

(Abrarns et al.. 1993: White et al.. 1994).

For germline clone analysis. the FRT (FLP ocombinase mget)/FLP-recombinase system

was used to induce mitotic recombination in developing FRT ~ [ o v o ~ ' ] (dominant female sterile

mutation)/FRTDaktlq; heat-shock FLP-recombinase female larvae. Unless a large homozygous

Dokrlq (non-~[oro~'r) clone is made in the ovaries. the resulting fernales will be sterile (Chou

and Pemmon. 1996). Thus, the FRT P [ ~ V ~ ~ ~ ] / F R T D ~ ~ ~ I ~ : hsFLP fernales c m only produce

embryos (hereafter referred to as GLC embryos) that lack the materna1 contribution of DAktl.

Dakrlq GLC embryos show extensive and ubiquitous ectopic apoptosis begiming at stage 6 (3.5 8

hours AEL). Presumably, non-GLC Dakt IV homozygous mutant embryos have enough

matemally-derived DAktl to rescue them to 1st or 2nd instar larvae before dying. Prior to the

larval-Iethal stage of development. the requirement for zygotically-expressed D m 1 is masked

by the presence of the maternally-derived enzyme. It may be at this stage of development when

zygotic expression becomes absolutely necessary. possibly due to a gradua1 loss of the

matemally-derived enzyme during development (Staveley et al.. 1998).

Recently . organ-speci fic over-expression studies and clonal analy sis have s hown that

DAktl signaling is required for more than just ce11 survival in Drosophila. The Drosophilu

homologues of a mammalian PKB activation pathway which includes the insulin receptor. the

insulin rrceptor substrate (IRS). both subunits of PI-3K (DPI-3K). PKB (DAktl ) and p70S6

kinase positively regulate ce11 size (and ce11 proliferation in the case of DPI-X) in wing and eye

tissue (Bohni et al.. 1999: Chen et al.. 1996: Montagne et al.. 1999: Scanga et al.. 2000: Verdu et

al.. 1999: Wrinkove et al.. 1997: Weinkove et al.. 1999). The pathway is thought to affect ce11

size through the phosphorylation and activation of the S6 ribosomal protein that is believed to

increase translation of ribosome-specific proteins which Iead to an increase in general protein

synthesis and ce11 growth (Montagne et al.. 1999: Rameh and Cantley. 1999: Thomas and Hall.

1997). Drosophila PTEN (DPTEN) has been s h o w to have the opposite effect in these tissues

and acts as an effective inhibitor of DAktl pathway-induced cell size phenotypes (Gao et al..

2000; Goberdhan et al.. 1999: Huang et al.. 1999: Scanga et al.. 2000). Although these studies

have focused on ectopic gene expression or clonal analysis in specific tissues. it is interesting to

note that larvae. pupae and adults that are homozygous for mutations in genes that activate the

pathway are al1 considerably smaller than their wild-type counterparts (Bohni et al.. 1999: Chen

et al.. 1996: Scanga et al.. 2000: Weinkove et al.. 1997: Weinkove et al.. 1999). However. the

fact that these tiny mutants are normally-proportioned suggests that the DPI-3KIDAktl signaling 9

pathway is ubiquitously-expressed and universally essential for ce11 size regulation (Edgar'

1999).

The objective of my tesearch was to idrntify and characterize novel upstream or

downstream DAktl signaling pathway components. The recessive larval lethal Duktlq mutation

has provided a genetic tool For identifying novel signaling pathway components. A. S.

Manoukian and I developed a rrans-heterozygous enhancer of lethality screen and I used this

screen to identiQ Drosophila mutations that showed a genetic interaction with the DakrlY

mutation. Interacting mutations were considered to be in genes rncoding potential pathway

components and could then be studird in îùrther detail. Thus. the identification of novel

pathway components would make it possible to either further charactenze the anti-apoptotic and

ce11 s i x regdatory functions of the DAktl signaling pathway or to identify new roles for this

kinase during development. The results of this screen indicated a possible interaction between

DAktl and the bHLH-PAS transcription factor Trachealess (Trh). which is required for

development of the Drosophile tracheal system.

At stage 10 (1 hours after z g -y-AEL) of embryonic development. clusters of cells on

both sides of rach of the ten posterior parasegments (T',-AS) assume a tncheal-specific fate.

During stages 10 and 1 1 (4-5h AEL). each cluster of precursor cells undergoes two

postblastoderm mitoses: the first mitosis establishes a region in the ectoderm called the tracheal

placode. while the second mitosis is coincident with invagination of the tncheal placode and

formation of the tracheal pit. Subsequent formation of the tracheal system. which is an array of

multi-. uni-. and subcellular tubes. occurs by ce11 migration and ce11 shape changes without

further ce11 divisions (Figure 3) (Bate and blartinez-Arias. 1993: Samakovlis et al.. 1996).

Tracheation occurs in three morphologically-distinct stages: prirnary (multicellular) branching.

secondary (unicellular) branching and terminal (subcellular) branching. The pattern of primary 1 O

Figure 3-Tracheal system development

Tracheal System

Figure: Diagram matic representation of tracheal devel opment. By stage 1 1 the tracheal pits (tp) have begun to invagi nate. Throughout stages 12 to 17. these pits wil l branch out. migrate. fuse in some places wi th other adjacent branches and eventual ly initiate terminal tracheation to ~ a c h and supply O2 to al1 em bryonicAarva1 tissues (note: termi na1 tmcheation is not depicted). The stage 5 embryo depicts an early fate map for tracheal precurson. Dorsal is up and anterior to the left for a11 em bryos. Figure Reference: Atlas of Drosophila Development. CSHL Press, 1993

and secondary branching which initiates from the tracheal pit in each tracheal metamere is

stereotypical, but the pattern of terminal branching is highly variable and appears to be

dependent on the oxygen needs of target tissues (Jarecki et al.. 1999: Metzger and Krasnow.

1999). The entire tracheal network in the èmbryo is continuous and supplied by a single

opening. the posterior spiracle. which forms during stage 11. Although the tracheal system

develops during embryogenesis. it is only required after the embryo hatches. Embryonic

requirements for gas exchange are met by the passive diffusion of gases across the embryonic

membranes. In fact. the tracheal systrm is tilled with tluid throughout embryogenesis and this

tluid is not clrared until just prior to l a n d hatching(Bate and Martinez-Arias. 1993: Samakovlis

et al.. 1996).

Coincident with the establishment of tracheal ce11 fate at stage I O is the expression of the

transcription factor Trh in the tracheal precursor cells. Trh is a member of the bHLH-PAS

family of transcription factors. This 949 arnino acid protein contains a bHLH (basic hrlix-hop-

helix) DNA-binding domain. a PAS (homlogy to o r . &nt and Sim) target gene specificity

domain. a transcription activation domain as well as a nuclrar localization signal (NLS) (Figure

1). Trh-specific antibody staining has shown that the Trh protein is localized to the nuclei of the

tracheal cells and that once its expression is initiated it is maintained for the remainder of

embryogenesis and throughout most of larval development by an auto-regulatory feedback loop.

The Trh protein is required for the initiai invagination of the tracheal placodes. tubulogenesis and

the expression of tracheal-specific genes (Isaac and Andrew. 1996: Wilk et al.. 1996). The

segment polarity genes r v g (rvingless) and dpp (ckcapentaplegic) are required for the spatial

localization of the Trh signal within the parasegment. helping to define the dorso-ventral and

antero-posterior expression pattern of Trh. respectively (Figure 5) (Wilk et al.. 1996).

Figure &Structure of the Trh protein

bHLH PASA PAS B

t t t NLS

Consensus

Putative phosphory lation sites

RGRGRSA RSRLPSI

Figure: Diagnmmatic representation of the Trh protein. showi ng the basic Heli x-Loop-Heli x (bHLH). PAS A and B domai ns. the putative Nuclear Localization Signal (NLS) and the consensus phosphorylation sites at positions 565-571 and 660-666.

Figure 5-Trh signali ng pathway

Trh - Nuclear Localization

Btl FGF Receptor Tdf TF

Others (?)

Figure: A diagram matic representation of the Trh signal h g pathway . Dpp and Wg signal h g establ ish the initial spatial expression of Trh. Trh then heterodimerizes with Tgo. translocates to the nucleus and i ni tiates transcri ption of tracheal -specific genrs.

Trh signaling is dependent upon the heterodimerization of Trh with the ubiquitously-

expressed Drosophila Arnt (DAmt)/Tango (Tgo) gene product (Figure 5) (Ohshiro and Saigo.

1997: Sonnenfeld et al.. 1997: Wilk et al.. 1996). Tgo is also a bHLH-PAS protein and is the

homologue of the mammalian protein ,4mt. which also functions as a dimerization partner for

transcription factors in mammals (Sonnenfeld et al.. 1997). Trh must heterodimerize with Tgo in

order to translocatr to the nucleus and activate transcription of nuclear target genes. including

itsrlf (Ohshiro and Saigo. 1997: Sonnenfeld et al.. 1997: Wilk et al.. 1996). Full Trh activity

requires the initiation of a transcriptional auto-regdatory feedback loop that amplifies the low

level of primary Trh expression to a higher. functional level (Wilk et al.. 1996). This feedback

loop also maintains Trh expression throughout development (Wilk et al.. 1996). Once Trh

expression has been arnplified. the Trh::Tgo transcriptional activator complex can recognize the

cis-regulatory CNS rnidline elrment (CME) in the target genes that are destined For tracheal-

specific transcription (Ohshiro and Saigo. 1997: Sonnenfeld et al.. 1997: Wilk et al.. 1996).

Trh is required for the expression of tracheal-sprcific genes such as the Breïrthless ( B d )

FGF receptor and the rracheoe deficriw ( t c f l transcription factor (Eulenberg and Schuh. 1997:

Wilk et al.. 1996). During the rarliest trachcal development (primary branching). the Btl

receptor binds the spatially-restricted Branchless (Bnl) FGF ligand and initiates the non-

directionally-speci fic. c hemotactic outgrowth of the p n m q trac heal branches towards the sites

of highest Bnl expression (Figure 6) (Klambt et al.. 1992: Lee et al.. 1996: Sutherland et al..

1996). B tPE3nI-deprndent branch outgrowth is the general mechanism required for tracheal ce11

migration events but is considered non-directionally specific because the placement of the Bnl-

expressing sub-epidermal mesrnchymal cells and sensitization of the tracheal cells to specific

branch fates are determined by other mechanisms (Bier et al.. 1990; Vincent et al.. 1997: 15

Wappner et al., 1997). Secondary branching and O?-sensing terminal tracheation is thought to

occur by a similar BtllBnl-dependent mechanisrn of chernotactic outgrowth (Jarecki et al.. 1999;

Metzger and Krasnow. 1999). The Tdf transcription factor is required for general ce11 migration

events during tracheal deveiopment and is thought to act in a parallel pathway independent of the

BtllBnl FGF signaling pathway (Eulenberg and Schuh. 1997).

Examination of the Trh amino acid sequence revealcd two putative PKB phosphoqlation

sites located at positions 565-571 and 660-666. on either side of the nuclear localization

sequencr (Figure 4). The recognition sequencr for PKB phosphorylation (reported previously

as Arg-X-Arg-Y-2-Srr/Th-Hyd) was conserved at each of the two sites: site 565-571 was Arg-

GIy-Arg-Gly-Arp-Ser-Ala and the sequence at site 660-666 was Are-Ser-Arg-Leu-Pro-Ser-IIe.

(Note that the Gly residue at position 568 of the tirst site was not in accordance with the

consensus.) Jing Jin of Dr. J. R. Woodgett's lab demonstrated that the second site was indeed

the preferred site for phosphorylation by mammalian PKB in riîro (unpublished results). This

was done by creating Ser to .41a point mutations in the Trh protein at Sed70 and Ser665. the

proposed target residues for phosphorylation. These point mutations would prevent

phosphorylation at those sites in an in vitro kinase assay. Jing Jin showed that wild-type Trh was

an excellent phosphorylation rarget of PKB. However. the single mutant. Ser665Ala. and the

double mutant. Ser570Ala/Ser665Ala. were not phosphorylation targets. while the single mutant

protein. Ser57OAla. was a phosphorylation target.

Since our screen implicated an in vivo interaction between DAktl and Trh. and since Trh

is a direct phosphorylation target of PKB in vitro. mp objective was to characterize the in vivo

interaction between these proteins and determine the role of DAktl signaling in the Trh-

dependent tubulogenesis pathway.

Figure 6-FGF receptor-dependent signal ing during tracheal branch formation

DTa DTa J

VB LT LTa LTp

Earl y tracheal ce11 migration Late stage tracheal metarnere . = tracheal ce11 s

a = mesenchy mal cells

=diffûsingFGFligand

DB: Dorsal Branch DT: Dorsal Tmnk VB: Viscerd Branch LT: Lateral Trunk

Figure: Figure 6 is a diagrm showing Btl/Bnl FGF signding in the tracheal cells. To the le fi. the Bnl li gand is expressed by mesodermai ce11 s and then di ffuses through the extracel lu1 ar matrix towards the Btl-expressi ng tracheal ce1 1s. Cr1 l m igntion and ce11 shape changes occur during the chernotactic outgrowth and branching of the tracheal ce1 1s towards the Bnl -expressing ce1 1s. To the right is a di agrm depi cting the general branc h structure of eac h tracheal me tamere. The DT anterior and DT posterior are hsed to their DT counterparts in adjacent tracheal metameres and the LT has branched to form the LT anterior and LT posterior.

2. Materials and Methods

2.1 Fly Stocks and Genetic Analysis

Drosophiln melanoguster were raised on media containine 21.74 g yeast. 65.22 g

commeal. 12.83 g sugar. 9.35 g agar. 21.73 g mL molasses. 13.06 mL teçosept solution. 2 mL

propionic acid and 0.1 g ampicillin per 1 L. Flies were maintained in either vials or boales at

roorn temperature (19-27°C) or 2j°C. A list of al1 stock lincs used in this study is summarized in

Table 2. The trh? DoktlY. D p l l O' and p60.' mutants have bren describrd previously (Isaac

and Andrew. 1996: Staveley et al.. 1998: Weinkove et al.. 1999: Wilk et al.. 1996). Unless

otherwise indicated. embryos were counted and the phenotypes reflected the expected

frequencies. according to genotype.

For each cross in the Daktlq trcrm-heterozygous enhancer of lethality screen. yiv:

FRT(2.1S7B)Daktlq. Tm3. Sb ['rr LmZ] females were crossed to wild-type males and the

resulting FRTL?.482B)Dakrlq- virgin female progeny were collected. Each heterozygous viqin

was then crossed to a P element-induced mutation that mapped to either the second or third

chromosome. Embryos from this cross were collected in vials for 8-10 hours at 25°C.

transferred in groups of 5 to a fresh apple juicc-agar media plate (3.4% aga. 50% apple juice).

and allowed to complete embryogenesis overnight at 25°C. The following day. embryos were

scored for lethality.

For Daktlq mutant analysis. ernbryos were double stained with anti-Trachealess and anti-

P-Ga1 antibody (described below) in order to accurately mark non-mutants. For brearhless in

sirri hy bridizations and MAb2A 12 stainings in Dakt IV mutants. yrf; FRT(2rl82B) DaktlqiTm3. Sb

[eveLucZ] females were crossed to wild-type males and the resulting non-Sb male progeny were

used for embryo collections with p v ; FRT(2;182B)Dukilq/Tm3. Sb[eveLacZ] virgin females in

order to avoid Tm3. Sb[eveLacZ] homozygous embryos.

S imilarly , for the over-expression of rrhBN I 7 in a Dakt14 mutant bac kground. w;

~ " . - 1 ~ ~ r h ~ ' ~ ' : FRT(2.4B2B)DokiZq~Tm3, Sb females were crossed to w: C . - I S T ~ ~ ~ ~ ~ " ; KiJm3. Sb

males and the irl: L L ~ S T ~ ~ ~ " ' ~ ; FRT(2;182B)Drrkrl?'E;i male progeny were then crossed to ilJ;

nrrnadillo (arm) GA L4; FRT/2A82B)Dukt14/ Tm3. Sb virgins for embryo collections.

For rescue of the rrh'JJ3 mutant. iiJ; 0rmGAL-l; irhiJS3, Tm3 females were crossed to either

iv: ~ + 4 ~ ~ r h ~ ~ ' " ; trh 'Js' Tm3 or iv; ~ ! î ~ ~ r h ' " ~ " " " , rrh'J83r'~m3 males.

The P element-induced Dukrl mutation. PM?-. was tested for complementation with

Dakilq by crossing p v : FR T(2.482Bj Dukilq; Tm3. Sb[eveLocZ] female virgins to Pl 67 7/Tm3. Sb

males and scoring for non-Sb adult progeny. To contirm that the P element mutant is nllelic to

Dakri. P i6X;Trnj. Sb males were crossed to w; L'C'o; Ki/Tm3. Sb females and then Cyo:

P1627Tm3. Sb male progeny were crossed to rither i v ; L'.dSDAkiI/Cyo: Ki/Tm3. Sb or i v :

armG.4 L4; f(i: Tm3. Sb. O f the resulting progeny. rrrniG.4 L4ICyo; P I 6 P T m 3 . Sb males were

crossed to L!4SD.-fkrl/Cyo: P 162 7Tm3. Sb fernales. To analyze the zygotic expression pattern

of DAkt 1. as assayed by P-Ga1 expression from the P1627Tm3. Sb P element insert. embryo

collections were taken from cups of wild-type virgin females crossed to Pl6?7/Tm3, Sb males.

For heat-shock experiments with hsp 7UGA L4/Ci4STrh Ser665.4 Iu embryos. 0-3 hour embryos

were aged for 1 hour and then heat-shocked for 9 minutes in a 37°C water bath just prior to

fixation.

Table 2: List of stock lines used in this study

Stock Lines

OregonR (wild type)

\v 1118

iv: L/CyO: ~ i f i z "?Tm3 , Sb

rv: armadillo (arm) GA L4; Ki/ Tm3, Sb

w : hsp XGA L 4 (2"')

w: pnired(prd) GA L4 (2"')

pv ~ s F L ~ ' ; C.vD/Tm3. Sb

p v ~sFLP"; ~ ~ ~ ( 2 . 4 8 2 ~ ) ~ [ o v o ~ ~ ] / T n i 3 . Sb (males)

i t l : U S P K B : LyTm6B

w; U;1SDakil!CyO; KiiTm3. Sb

ii*: armGA L4; FR T(2~82B)Dokt I q Tm3. Sb

kt7: FR T(?A 82 B) Duki 1 Y Tm3. Sb

rv: LASDPTEN (7"")

DPTEV~~' CYO

P 1 74 7(trh)/Tm3, Sb

PI6ETm3, Sb

ALI P element lines for the Dakt h c r e e n

rv : FR T(2.4 82 B) P l6DTm.3, Sb

iv; p60"43c, Gla. bn; Pg[R 1 O]

iv: Pg[H]; Dp I l 04iTrn3

1s; irh'J83/~m3, Sb

JW; FRT(Z.1SZB)Dakrlq~Tm3. Sb[eveLncZ]

IF: LX ~ T r h ~ ' " ' (-rJ)

iv ; L X S T ~ ~ ~ ~ " ~ Ki/Tmj. Sb w; uASTrh?3t?r66~.~b 7"d

(- )

><,,. ~ ~ s ~ ~ h s ~ r 6 6 " ~ a . , Kiflm3. Sb

ir; ~ ! 4 ~ ~ r h ~ . ' * ~ (jrd)

iv: ~:-1~Trh~"': rrh'J83rTm3. Sb

iv; UA ~ ~ r h ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ : rrh 7J83/~m3. Sb

rv; ~ ' , 4 ~ ~ r h ~ " " ' ; FRT(2A82B)Dakrlq:Tm3. Sb

Source

A. S. Manoukian

A.S. Manoukian

A.S. Manoukian

A S . Manoukian

A.S. Manoukian

A S . Manoukian

A.S. Manoukian

A S . Manoukian

A.S. Manoukian

A.S. Manoukian

A.S. Manoukian

A S , blmoukian

A.S. Manoukian

A.S. Manoukian

Bloomington Stock Center

Bloomington Stock Center

Bloomington Stock Center

R. Binari

Weinkove et al. ( 1999)

Weinkove et al. ( 1999)

Isaac et al. (1996)

B. Wetsc h

B. Wetsch/ N. Anthopoulos

B. Wetsc h

B. WetscW N. Anthopoulos

B. Wetsch

B. Wetschi N. Anthopoulos

B. Wetsc h

B. Wetsch

B. Wetsch

2.2 Large Sample Confidence Interval for n Population Proportion

To determine the confidence intemal for the true mêan of the population as estimated by

the proportion of successes (expressed as a percentaçe) in the sample of embryos that were

scored for each cross. 1 used the equation:

iv ; armGI1 L-l; trh'"3/~m3, Sb

w; L2SDaktl/CyO; L L ~ s T ~ ~ ~ . ' ' ~ '

i v ; UASPKB: ~ / . 4 s ~ r h Bs'9

where p is the percentage of successes (lethal embryos) that were scored in the sample embryos

tiom each cross and n is the number of embryos scored for each sarnple. The critical value z was

B. Wetsch

B. Wetsch

B. Wetsch

set at 1.960 so ihat there was a 95% confidence levrl that the true population mean lies between

the expressed values. The confidence interval for the truc population mean of each samplr t a s :

wild-type cross (0.0. 2.1). Daktlq cross (0.3.2.7). P l -4: cross (0.2. 2.4). Dpll~': Pg[H] cross

(0.0.1.6), ~~60'; Pg[RIO] (0.0. 2.1). DaktlY x Pi-4: cross (4.1. 8.9). Daktlq x ~~1 10'; Pg[H]

cross (1 .I. 3.6) and Dakilq .u ~ ~ 6 0 ~ ; Pg[RlO] cross (8.6. 16.4).

If the values for the confidence intervals from two different samples overlap then the

difference between the population rneans is not significant: however. if the confidence intervals

do not overlap then it can be stated with 95% confidence that the difference between the

population means is significant. A summary of the cornparisons that were made for the

confidence interval from each sample of embryos is presented in Table 3. 'Significant' means

that the difference in lethality that was observed between the two sarnples was found to be

significantly different and Wot Significant' means that the difference in lethality that was

2 1

obsented between those sarnples was not different enough to be considered significant at a 95%

confidence interval. Note that cornparisons were only made between experimental crosses and

their relevant control crosses; thus. NIA (Not Applicable) signifies that the cornparison was not

made because it was not meaningful or necessary.

Table 3-Confidence Interval Analysis for the Daktlq Tram-heterozygous Enhancer of Lethality

Screen

Wild-ty pe

Daktlq x 1 Significant 1 Significant 1 Significant / NIA 1 NIA

cross

cross N A

1 Significant 1 Significant 1 Significant 1 Significant

PI 74 7 Daktlq x

Dp11D4 / Significant 1 Significant 1

cross Not

LIp6@ Daktlq x

2.3 Cuticle Preparations

Significant

Following a collection on apple juice-agar plates. embryos tvere plated ont0 a fresh plate

and aged ovemight at 2j°C. Unhatched embryos were dechorionated with 50% bleach. rinsed

cross Not

Not

with water. then mounted and cleared in Hoyer's medium for 24 hours before

Significant

analy sis/photography . Embryos were pho tographed under dark field to enhance cuticular

structures using a Leica DMLB microscope with Kodak TMAX ASA 100 black and white film.

cross Not

Not

2.1 Embryo Collection and Fixing

cross Not

NIA

Populations of Drosophiia used for embryo collections were generated by combining 150

1 N/A 1 NIA

fernales and 50 males of specific genotypes in a collection cup containing a lightly-yeasted apple

Signi ficant

Not

juice-agar (3.4% agar. 50% apple juice) media plate. Collection times varied in order to generate

N/ A

embryos at specific developmental stages. Afier collection. embryos were fixed as described

(Manoukian and Krause. 1997). Embryos were gently removed from collection plates using a

brush. collected in a vial with a nylon mesh bottom and washed several times with water. The

outer chorion shell was removed from embryos by irnrnersing the vial in a freshly-prepared 50%

blrach solution until the dechorionatrd embryos could be seen floating near the surface (1-2

min). Dechorionated embryos w r e washed extensively with water and placed in a scintillation

vial containing a freshly-prepared 2-phase solution of formaldehyde/PBS (680 pL 37%

formaldehyde in 5 mL PBS (1.44 g Na2HP04. 8 g NaCI. 0.2 g KC1.0.24 g KH2PO~ per 1 L. pH

7.2) and heptane. Embryos were tked for 20 minutes. afrer which they were removed and

transferred to a 1.jrnL eppendorf tube containing 500 pL of heptane and 500 pL of 100%

methanol. This ?-phase mixture was vortexed for 1 minute to rernove the vitelline membrane.

The upper heptane phase was drawn off and the embryos were subsequently rinsed 7-8 times in

ImL of 100% methanol before being stored at -20°C (in methanol).

2.5 Antibody Staining

For visualization of antibody staining in embryos. the alkaline phosphatase color reaction

was used. Fixed embryos were re-hydrated in a 0.5 mL èppendorf tube with 7-8 rinses of TBST

(140 m M NaCl. 50 mM Tris-HC1 pH 7.4. 0.05% Tween-20. 0.05% TritonX-100). Embryos

were washed 3 times in TBST for 20 minutes per wash on an orbital shaker and then incubated

ovemight at 4OC with a pnmary antibody that \vas diluted in TBST (Table 4). Embryos were

washed 3 times with TBST for 20 minutes each and subsequently incubated for 1.5 hours at

room temperature with the appropnate biotinylated secondary antibody (Vector Laboratories)

that had been diluted 1:300 in TBST. Five 30-minute washes in TBST followed. A final

incubation using alkaline phosphatase-conjugated Streptavidin (diluted 1300 in TBST) t-as done

at room temperature for 45 minutes. The embryos were washed 4 times for 20 minutes with

TBST and rinsed 2 times in alkaline phosphatase buffer (AP buffer; 100 mM NaCl. 50 m M

MgCl?, 100 mM Tris pH 9.5. 0.1 % Tween-20). The color reaction was initiated by adding

freshly-prepûred reaction mix (4.5 PL nitro blue tetrazolium (NBT) and 3.5 pL 5-bromo-4-

chloro-3-indolyl phosphate (BCIP) in 1 mL AP buffer). Once the signal developed to a deep

purple, the reaction was stopped with 5-6 washes of TBST. Excess liquid was removed and

lOOuL of embryo mountant (70% glycerol. 30% 100 mhl Tris pH 8.0) was added. Embryos

were allowed to settle to the bottom of the tube be fore being transferred ont0 microscope slides

for observation. A Leica DMLB microscope containing Nomarski optics was used to

photograph stained embryos at 20x magnification with Kodak Ektachrome 64T color reversa1

film.

Table 4-Primary antibodies used in this study

Antibody

polyclonal anti-Trac healess

monoclonal MAb2A 1 2

monoclonal anti-P Ga1

monoclonal anti-DIG(AP)

monoclonal anti-FLAG

--

Dilution I Source

2.6 Double Antibody Staining

1:300

120

1:lOOO

1 :2000

1:SOO

For Anti-Trachealess staining in embryos from a p v ; FRT(2A32B)Dakt l 4 Tmj[eveLacZ]

(Isaac and Andrew, 1996)

(Sarnakovlis et al.. 1996)

Boehringer Mannheim

Boehringer Mannheim

Sigma- Aldrich

collection cup. double antibody staining was used to distinguish mutant heterozygotes and

balancer homozvgotes from Daktlq mutant homozygotes. The sarne protocol used for antibody

staining (described above) was used for double stainings; however. the primary antibodies (anti-

Trh and anti-e-Gal) were mixed (1 :300 anti-Trh into 1:3000 anti-B-Gal) for the first antibody

incubation. Washes were done as described and then each biotinylated secondary antibody was

t 4

independently incubated for 1.5 hours with two 30-minute washes between each antibody

incubation. The postsecondary antibody washes. 45-minute Streptavidin incubation. final

washes and development of the signal were done as described in section 2.4.

2.7 PCRIDIG-Labeled Probe Synthesis

The PCR1digoxigenin (D1G)-labeled DNA probe synthesis protocol and stock solutions

were obtained from Boehringer Mannheim. The procedure was done as described by N. Patel

and C. Goodman in the Boehringer Mannheim catalogue. A DIG-labeled Btl probe was made

from a 2.3Kb BtI cDNA in pBlueScript (donated by D. Isaac) that was Iinearized with a

restriction digest at a unique BglII restriction site in the multiple cloning site. The following

reaction was set up in a 0.5 mL eppendorf tube: 3.5 PL 1Ox PCR reaction mixture. 2.5 pL DIG

DNA labrling mixture. 5.0 pL T3 primer (5.4 PM) and linearized plasmid DNA to a

concentration of 400 ng/pL. The mixture was brought to 25 pL with dH20 and thoroughly

mixed. Forty pL of mineral oil was layered on top of the mixture. The eppendorf tube was

placed in the PCR thermal cycler (PTC-100 Programmable Thermal Cycler, MJ Research Inc.)

and the program began as follows: 95°C for 3 minutes. 88OC for 3 minutes. Two pL of Taq

DNA polymerase (1 2 5 U total of Taq polyrnerase. made by 1:8 dilution in dH?O of j U / l L Taq

polymerase stock. Boehringer Mannheim) were added during the 88°C incubation. This

Taq/primedplasmid mixture was incubated for 34 cycles under the following conditions: 94OC

for 45 seconds. 60°C for 43 seconds and 72°C for 2 minutes. The PCR run was followed by a

IO-minute incubation at 72°C. Following the PCR reaction. 75 pL dHzO were added below the

mineral oil layer and 95 pL of the PCR reaction mixture was removed. DNA was precipitated

by adding 2.0 PL 5M NaCl. 1.0 pL E. coli tRNA ( IO mg/mL), 0.5 yL glycogen (20 mg/mL) and

300 pL 100% ethanol. The mixture was vigorously mixed and placed at -20°C ovemight to

allow DNA to precipitate. The mixture was then spun at 14000 rpm for 30 minutes at room

temperature in an Eppendorf centrifuge Mode1 541 5 C. The pellet was washed once with 70%

ethanol. air-dried at room temperature and resuspendcd in 200 pL hybridization buffer (50%

deionized formamide, 5X SSC. 100 pg/mL sonicated and boiled salmon spem DNA. 100 pg/mL

tRNA, 50 pg/mL heparin and 0.1% Tween-20). Before storing at -20°C. the DIG-labeled DNA

probe was boiled for 1 hour.

2.8 Whole-Mount Na Situ Hybridization

In sitir hybridization to whole mount embryos was perfomed as described (Manoukian.

1997). The alkaline phosphatase color reaction was used for visualizing DNA probe

hybridization in embryos. Fixed embryos were re-hydrated in a 1.5 mL eppendorf tube with 2

rinses of 50% methanoll 50% PBT (1 x PBS. O. 1% Tween-30) and then 5-6 rinses of PBT. The

embryos were fixed for 20 minutes with 5% formaldehyde in PBT and then washed 3 times for 5

minutes in PBT. Embryos were incubated in 50 pdmL proteinase K in PBT For 2 minutes.

Incubating in 2 mg/mL glycine in PBT for 5 minutes stopped the reaction. Embryos were

washed 2 X 2 minutes with PBT. again fixed for 20 minutes in 5% formaldehyde in PBT and

then washed 5 X 2 minutes in PBT. At this point the embryos were transferred into a 0.5 mL

eppendorf tube. washed in 50% PBT 1 50% hybridization solution and then in 100%

hybridization solution. Embryos were prehybridized for 1-2 hours at 48OC in 100 pL

hybridization buffer. Meanwhile. the DIG-labeled DNA probe was diluted in hybridization

buffer (generally 1 :IO), boiled for 5 minutes. chilled in an ethanoll dry ice bath for 1 minute and

equilibrated to 48°C for 1 minute brfore being added to embryos. Hybridization occurred

ovemight at 48OC. Following hybridization. embryos were washed at 48°C for 20 minutes in

400 pL hybridization buffer. 400 pL 50% hybridization buffer / 50% PBT and 5 times in I mL

PBT. Embryos were incubated with an anti-DIG alkaline phosphatase-conjugated antibody

(diluted 1:2000; Boehringer Mannheim) for 2 hours at room temperatiire while shaking. Five

20-minute washes in PBT at room temperature were followed by two 1-minute washes in AP

reaction buffer (100 mM NaCl. 50 m M MgCl?, 100 mM Tris pH 9.5. 0.1% Tween-20). The AP

reaction was developed to a deep purple color afier which the embryos were washed 5-6 times

with PBT to stop the reaction before mountant was added. Once embryos settled. they were

transferred onto microscope slides and photographed as described in section 2.4.

2.9 Confocal fluorescence microscopy

Fixed embryos were treated as in Section 2.4 (brightfield antibody stainings). with only

minor differences. After re-hydration. embryos were incubated for two hours at room

temperature. while shaking. in Block TBST ( 1 xTBST with 0.5% Milk powder and 0.25% BSA).

Al1 subsequrnt washes following primary antibody incubation. secondary antibody incubation

and Streptavidin incubation were done using Block TBST. After the addition of light-sensitive

FITC-labeled Streptavidin ( 1 300 dilution). al1 subsequent steps were done in the dark. Embryos

were mounted using the "Molecular Probes" SlowFade Light Antifade Kit. Embryos were tirst

equilibrated for 5 minutes using the SlowFade equilibration buffer. The buffer was removed and

the embryos were mounted in lOOuL of SlowFade mountant [Reagent A: Glycerol (1: l)].

Analysis of embryos was done using the Zeiss Confocal Microscope-LSM 510 at 63x

rnagnification with an excitation laser of wavelength -160-480nM from an Argon laser.

Computer-scanned images were stored on compact disc.

2.10 Germ-Line Clone Generation

Generation of germ-line clones (GLCs) using FRTIFLP targeted recombination was

performed as described in Chou et al. (1993). w: FRT(2.482B) P1627/Tm3. Sb fernales were

crossed to J W ~ S F L ~ ' ; FRT(82B) ~ [ o v o ~ ' ] / ~ r n 3 males. The cross was transferred to a new vial 27

every day once eggs were deposited. Third-instar larvae were heat shocked at 37°C for 2 hours

on two consecutive days to activate transcription of the yeast FLP" recombinase. Larvae were

then transferred to bottles until adults eclosed. Virgin females containing germ-line clones were

crossed to iv; FRT(2dS3B) P1627;Tm3 males and the resulting GLC embryos were analyzed

under dark-field microscopy for perturbations of the normal cuticular pattern.

2.1 1 Bacterial Transformation of Btl cDNA-Containing Plasmid

DHSa competent cells were thawed on ice before addition of 1-Zpg plasmid DNA,

followed by gentle mixing and Further incubation on ice for 30 minutes. Crlls were incubated at

42°C for 30 seconds and then incubatrd for 2 minutes on ice. The cells were then grown in a

rotary shaker at 37°C for 45 minutes in jOOpL of LB. A SOpL aliquot was plated ont0 a fresh

LB-Ampicillin plate and gram ovemight at 37OC. Single colonies were then restreaked ont0

another LB-Amp plate. grown overnight at 37OC and stored at 4°C.

2.12 Mari Prepnration of Btl cDNA-Containing Plasmid

A single colony from a freshly-streaked LB-amp plate was used to inoculate a starter

culture of 2 mL LB medium containing 50 pg/mL ampicillin. The culture was incubated

overnight at 37°C with vigorous shaking (224 rpm). One mL of the starter culture was used to

inoculate 200 mL LB medium which was again incubated at 37°C overnight with vigorous

shaking. Bactenal cells were collected in 50 rnL Coming tubes by centrifugation at 5000 rpm

for 15 minutes in an eppendorf centrifuge Mode1 5804 R. Plasmid DNA was isolated using the

Qiagen Qiafilter Mêuiprep Kit following the manufacturer's instructions. The yield and purity of

the DNA was determined by UV spectrophotometry.

2.13 Embryo Microinjections

2.13.1 DNA preparation

pL!AST[w*]-Trh DNA was purified using the maxi-prep protocol. The DNA was further

prepared for microinjection with phenol/chloro f o m extractions to remove impurities. For each

successive extraction. 400 yL of phenol, phenol: chloroform mix and chloroform was added to

the DNA. The two-phase mix was vortexed, spun in a microcentrifuge and then the upper phase

vas removed for the next extraction. Following the final chloroform extraction. the upper phase

was transferred to a new 1.5 mL eppendorf tube and the optical density at 260 and 380 nm was

measured to determine the purity and concentration of the DNA. Only when the 260/280 nm

ratio was between 1.7 and 1.9 was the DNA used for rmbryo injections. Ten pg of

pLXST[w']Trh vector was combined with 2 pg of phsir helper plasmid canying the transposase

eene. The volume was brought up to JO0 pL with additional TE and DNA was precipitated by C

the addition of 1/10 volume 3M NaOAc. pH 5.4 and I mL 100% cthanol. followed by mixing

and spinning for 30 minutes at 14000 rpm at room temperature. The dried pellet was then

resuspended in 20 PL of injection buffer (5 mM KCI. 100 mM Na2HPO4). Irnmediately before

microinjections. the DNA mixture was spun at 14000 rpm for 2 minutes before 2.5 PL was

d r a i n from the top of' the mixture using an Original Eppendorf MicroloaderC3 and loaded into a

pulled-glass injection needle (Original Eppendorf Femtotipa injection needle).

3.13 $3 Embryo preparation and injection

Collection cups containing w"" nies were set up with apple juicehgar plates that were

changed every hour for 2-3 hours prior to collections for microinjection. Embryos were

collected for 1 hour. dechorionated with 50% bleach. rinsed with water and lined up close to the

rdge of an agarose pad (2Y4 agarose in dHzO. with a small amount of dye for contrast) with the

postecior end at the edge. Embryos were placed on a slide containing double-sided tape and

immediately put in a desiccation chamber containing Dri-Rite for 7-1 5 minutes depending on

environmental humidity. Following desiccation, the embryos were covered with a thin layer of

heavy (Series 700) Halocarbon oil and the slide was placed on the stage of the injection

microscope (Leica LEITZ DM IL). Small amounts of the injection mix were deposited into the

posterior end of each uncellularized embryo in the area of the developing pole cells (N.

Anthopoulos graciously perforrned the injections). Cellularized embryos were destroyed.

Following injection. the embryos were surrounded by Vaseline petroleum jelly to create a well

and then covered with light (Series 56) Halocarbon oil. Slides were then placed on top of rnoist

Whatman paper in a large Petri dish. Petri dishes were stored in sealed plastic containers

containing moist paper towels and allowed to develop at room temperature for 36-48 hours.

Surviving larvae were picked out of the oil and transfened to vials containing Drosophilu media.

Approximately 25-30 larvae were placed in each vial. which was kept moist by small additions

of H20 . Adult survivors. the Go generation. were then treated as described in the following

section to determine the chromosomal location and number of p15fST[iv-]-Trh inserts for each

transformant line.

2.14 Mapping and Balancing Transformants

All Go adult survivors were rnated individually to rv"" females or males. From these

crosses. transformants were identified by screening for loss of white eye color since the

pUilST[iv-] vector carries a iv* marker. Al1 transformants (example: iv; pUST[rv']-TrW)

were treated individually and mapped accordingly . iv: p U,-4ST[w '1- TrM- females were mated to

w; L CyO; ~i fi^"'^ :Th13 males. From this cross. iv; pUAST[ic - 1-TrhKyO. TlW3 males were

mated back to w; LiCyO; ~ i f t ? " . Th13 females. The progeny of this cross were examined to

determine whether the pbWT[ i r .~ ] -Trh insert was on the second or third chromosome. An

insertion on the second chromosome yielded white-eyed progeny that were always I V ; UCyO and

orange-ey ed progeny that were either iv: ~ V * / C ~ O ; ~ifr-."'O/~m3 or iv: iv7L; ~i fizw24.~m3. An 3 O

insertion on the third chromosome produced white-eyed progeny that were always ~i /rz"'* /Th43

and orange-eyed adults that were either rv; L/CyO; w7Tm3 or iv; L/CyO; w-/~i jz \" ' " ' . In the

absence of a lethal insertion, the balanced and mapped transformant line was crossed inter se to

create a homozygous stock.

2.15 Western Blot AnaIysis

1.1 5.1 Protein Extraction

Zero ro 8-hour embryos were collected for each of the appropriate genotypes. flash frozen

on dry icelethanol and stored at -70°C. RIPA lysis buffer (5OmM Tris Cl [pH 7.51, l5OmM

NaCl. 1% Nonidet P-40. 0.5% sodium deosycholate and 0.1% SDS) was prepared with 1 pellet

of Borhringer Mannheim protease inhibitor in 7mL of buffer. On ice. a 2OOuL volume of

embryos was homogenized in a 7mL glass homogenizer in 5OOuL of RiPA lysis buffer. On ice.

the Iysate was collected into a 1.5mL eppendorfand cellular debris was cleared by spinning for 1

minute at l4000rpm in an Eppendorf centrifuge. Mode1 5414 C. On ice. 5OOuL of the cleared

Iysate was collected and lOOuL of 5xSDS loading buffer was added. Lastly. ten 5OuL aliquots

were boiled for 9 minutes. flash frozen in dry ice/ethanol and stored at -70°C.

1.15.2 SDS Gel Electrophoresis

Equal 40uL aliquots of each of the samples. plus ZOuL of a calibnted molecular weight

protein standard (Kaleidoscope Prestained Standards). were run in adjacent lanes on a 4%/6%

stacking/separating SDSRAGE gel (see Table 5) with lxSDS Gel Running Buffer (2.9g Tris

Base. 14.Jg Glycine and lg SDS in 1 L of dH20). The gel was mn at l8OV for 1 hour.

Table 5-SDS gel reagents

1 .SM Tris-HCL (pH8.8)

O.5M Tris-HCL (pH6.8)

10% APS

10% SDS

Reagents

40% AcrylIBisacryl( 1 9: 1 )

dHzO

TEMED

Stacking gel

0.25mL

1.6mL

2.5uL

2.5mL Total

Separating gel

2.15.; Westem Blot Transfer and Staining

1) Westem Transfer

Proteins were transferred to PVDF membrane by electroblotting. Before transfer.

blotting filter papers were thoroughly soaked with transfer buffer (25mM Tris-HCL. 192mM

Glycine and 20% Methanol: pH8.3) and the PVDF membrane was rinsed in mrthanol and then in

dHzO. A 'Blotting Sandwich' kvas prepared as follows: five filter papen were placed on the

basekathode. the gel was laid over the filter papers. the transfer membrane was laid against the

gel (being careful to exclude any bubbles that might interfrre with transfer). an upper layer of

ftlter papers ( 5 ) were Iayered on top and finally the lid/anode was securely screwed down on top

of the 'sandwich'. The electrotransfer was run at 10- 14V for two hours at 400mA.

II) Westem Blot Staining

The blot was blocked for two hours. while shaking. at room temperature in 1xTBST (140

mM NaCl. 50 rnM Tris-HCl: pH 7.4.0.05% Tween-îO.O.OS% TritonX-100) with 2% BSA. The

blot was then incubated in Anti-FLAG pnrnary antibody (1 5.000). while shaking. ovemight at

J°C in 1xTBST with 0.5% BSA. The primary antibody was removed and the blot was washed 4

times for 15 minutes in lxTBST with 2% Carnation milk. The secondary antibody, H M -

conjugated anti-mouse IgG (diluted 1 :IO. 000 in lxTBST with 0.5% BSA). was added and

incubated, while shaking, for two hours at room temperature. The secondary antibody was

removed and the blot was washed 8 times for 15 minutes in IxTBST with 2% milk. followed by

two rinses in 1xTBST. Enhanced Chemoluminrscence (ECL) was used to detect the signal.

Equal amounts of the ECL reagents (ImL total) were mixed. added to the blot and incubated for

2 minutes with vigorous shaking. The blot was patted dry and exposed to Kodak X-Omat Blue

autoradiography film.

3. Results

3.1 Daktlq Tram-Heteroygous Enhancer of Lethality Screen

I initiated a tram-heterozvgous enhancer of lethality screen. testing for the ability of P

element-induced mutations to cause significant ernbryonic lethality in Duktlq heterozygotes. P

elements are transposable genetic elements that generate mutations by site-specific insertion into

the genome and it has been shown that these mutations disrupt more than 25% of the estimated

3600 Drosophila genes that are vital for adult viability (Spndling et al.. 1999). In this screen.

heterozygous Daktlq;- virgin females were crossed to males from a single P element-induced

mutant stock that did not contain a Daktiq mutation. Assuming random and non-biased

segregation of the chromosomes. 25% of the embryos from this cross will be heterozygous for 2

mutations at different locations that are in trnns to one another: these are the trrins-heterozygous

embryos. Since the mothen from this cross are Driktiq heterozygotes. the DakrlY heterozygous

ernbryos from this cross lack half of the normal amount of both zvgotic and materna1 DAkt 1

kinase activity. Although the Daktlq heterozygous embryos are normally viable (showing only

1.5% embryonic lethality. Figure 7A). ~rans-heterozygous embryos are also missing one wild-

type copy of the gene represented by the P element-induced mutation. If the gene product

represented by this mutation normally has a function in DAktl signaling. it is possible that loss

of half of both the zygotically- and rnaternally-derived DAkt l kinase activity. when combined

with loss of half of the normal zygotic expression of the P element-induced mutation. could

result in an increase in embryonic lethality above the levels normally seen in wild-type embcyos

(Figure 7A). Cuticle phenotypes were analyzed to determine the function of each interacting

mutation within the DAkl signaling pathway. The results of this screen. s h o m in Table 6.

indicated a possible interaction benveen Daktlq and Pi 74 7. a P element-induced. non-embryonic

Figure 7-Dakrlq enhancer of lethality screen

Figure: Results from the tram-heterozygous enhancer of lethal ity screen usi ng the Daktlq mutant. The bar graph in Figure6A is a diagram matic representation of the lethal ity observed in control embryos and DaktIVmutant tram-heterozygous embryos. The nurnber of embryos (n) scored for each cross is: n (wild-type)=298. n (Dakt14 cross)=395. n (PI 747 cross)=379. n (Daki Iq x PI X ) = 4 l 8 . n (Dpll U4 cross)=190, n (Dakt14 x Dp I I Oi)= 594. n (Dp6W cross)= 2 16 and n (DaktP x Dp6U4)=272. Cuticle preparations for wild- type (Fi gure 6B). DaktPltrh(Pl74 7) (Figure 6C). and Dakt Plp6OA (Figure6D) are show.

lethal mutation of the bHLH-PAS transcription factor iracheaiess (rrh), a gene essential for

tubulogenesis during development of the DrosopMa tracheal system.

The rrans-heterozygous combination of Dakrlq/Pl 747 induced 6.5% embryonic lethality

(Figure 7A). 1 used confidence interval analysis to determine the significance of the difference

in embryonic lethality that was observed in the Daktlq x P I747 cross when compared to the

embryonic lethality that was observed in control crosses between wild-type parents. P 174 7

heterozygous parents or Dakrlq heterozygous parents. Using this statistical analysis. the lethality

that was observed in the Drzkr I q x P l 74 7 cross was shown to be significanrly different than the

lethality observed in each of the control crosses (see Table 3. Materials and Methods).

Assuming that the enhanced lethality (6.5%) is specifically induced in the truns-

heterozygous embryos. which represent 25% of ail embryos from the cross. an estirnated 26%

lethality (6.5%/25%) is being observed in the truns-heterozygotes. The PI -47 mutation is the

result of a P element insertion into the 5' untranslated region of the rruchroless (trh) gene that is

located at cytological position 6 1C of chromosome three. Analysis of the cuticle pattern of the

lethal DokilY/PI 7 4 7 trons-heterozygous embryos revealed mostly unhatched wild-type first-

instar larvae (Figure 7C) as well as an even smallcr number of embryos with non-specific

pleiotropic phenotypes (results not shown). The lack of an apoptotic phenotype suggested a

novel role for DAktl in a Trh-dependent developmental pathway that is separate from its role in

ce11 swival , which led me to further characterize the interaction between Dakt lq and PI 747

(rrh).

Table 6: Results from the DakîIg trans-heterozygous enhancer of lethality screen

Drosoplzila Stocks Used in Screen O h Lethality in Trans-

heterozygous embryos

2.0% of 558 Total

2.3% of 428 Total

3.0% of 976 Total

1.5% of 388 Total

1.4% of 1 42 Total

3.8% of 601 Total

0.8% of 134 Total

8.8% of 387 Total

6.9% of 607 Total

3.2% of 535 Total

2.4% of 594 Total

6.5% of 41 8 Total

2.7% of 334 Total

3.9% of 358 Total

3.0% of 364 Total

1.6% of 365 Total

4.4% of 183 Total

2.9% of 3 1 1 Total

3.2 Analysis of Trh Activity in Daktlq, and DPTEN"~" Mutants

Wild-type embryos were used to analyze normal Trh expression at stage 11 using the

anti-Trh antibody and to observe normal stage 1 I Br1 transcription (Figures 8A and B). In

addition. the completed structure of the vacheal system at stage 16 was visualized by staining

with the MAb2A12 antibody. which binds the tracheal lumen (Figure 8C) (Isaac and Andrew. 3 7

1996; Wilk et al., 1996).

Homozygous embryos of a strong. non-embryonic-lethal irh mutation. ~ h : ~ " , show

extremely weak and often undetectable Trh protein expression at stage 11 as revealed by

antibody staining experiments. Furthemore. embryos at stage 1 1 have no Btl transcription in in

sitir hybridization experiments: thus. there is no invagination of the tracheal precursor cells to

form the tracheal pits or any other tracheal structure (Figures 8D to F) (Isaac and Andrew. 1996:

Wilk et al., 1996).

Due to the large materna1 contribution to DAktl expression. it would seern necrssary to

generate nul1 expression Dtrkrlq GLC ernbryos in order to study the role of DAktl in Trh

signaling. However. as mentioned previously (Staveley et al.. 1998). Daktlq GLC embryos

begin to show extensive apoptosis and massive morphological defects just prior to the

developmental stage when Trh expression is normally initiated. This could make it very difficult

to determine whether there was a loss of Trh expression due to the loss of DAktl or whether the

effects on Trh expression were due to widespread cellular dysfunction and tissue loss.

Therefore. 1 analyzed the effect of the zygotic (non-GLC) Duktlq mutation on Trh

activity . Dakt l q homozygous embryos have enough matemally-derived DAkt 1 protein to rescue

most of the early zygotic expression requirements: thus. these mutants c m survive through

embryonic and early larval development. In fact. the partial loss of DAktl had no observable

effect on Trh expression in early stage 10 embryos. as assayed using anti-Trh antibody (results

not shown). However. nearly a third (28%) of the matemally-rescued Daktlq homozygous

mutant embryos had weak Trh expression dunng stage 1 1 when compared to wild-type embqos

at the same stage that had been stained using the sarne protocol (Figure 8G). Of the 32 late

stage 10 Daktlq homozygous embryos that were scored. 9 showved defects in Trh expression (see

Material and Methods for how Daktlq homozygous embryos were identified). Instead of 3 8

Figure 8-DAkt I signaling positively reçulates Trh activity

Figure: Genetic data showing that the DAktl signaling pathway positively regulates Trh

activity.

Wild-type Trh expression (as assayed by anti-Trh antibody staining). Trh nctivity (as

assayed by Bll il1 s i t ~ i hybridization) and trached structure (visualized using the

MAb2A12 antibody) are s h o w in Figures 8A-C, respectiveiy. Trh expression in the

~rli '~~',LIaktl~ and p60" mutant backgrounds is s h o w in Figures 8D. G and J.

respectively. In addition, B11 transcription in the ~ r l i " " . ~ u k t l ~ and p60" mutant

backgrounds is s h o w in Figures SE. H and K. respectively. Trxheal structure in the

~rh; ' " .~akr l~ and p60" mutant blickgroiinds is shown in Figures 8F. 1 and L.

respectively. The expression pattern of endogenous DAktl, as assayed by anti-B GAL

antibody staining in the P element-induced Düktl mutant, is shown in Figure 8M. Lastly,

the effect of DPTEN over-expression on Br1 transcription is shown in Figure 7N and the

effect of a loss of DPTEN is shown in Figure 70 . For dl embryos. anterior is to the left

and dorsal is up. Ernbryos photographed at 70x magnification.

affecting the earliest stage 10 Trh expression. partial loss of DAktl affects stage I l Trh

expression. This suggests that DAktl is not involved in the initial regulation of Trh expression

but is instead required for the later transcription-dependent auto-regulatory feedback mechanism.

The role of DAktl in Trh transcriptional activation was further studied by examining Btl

transcription in stage 1 1 Drrkrl%omozygotrs using in situ hybridization: in an estimated 30% of

the Daktlq homozygous embryos Btl transcription is weaker than in wild-type embryos (Figure

8H). Of the 104 embryos scored in the Dakr lq Br1 in situ experiment, 8 had weak Btl

transcription. Assuming the random and non-biased srgregation of the chromosomes from a

cross between DakrIY heterozygous parents. an estimated 26 (25% of the total) were D o k ~ l ~

homozygotes. Since the 8 affected embryos were assurned to be Daktlq homozygous embryos.

then 30% (8/26) of the Duktlq homozygotes showed Btl transcripton defects. No dekcts in Trh

expression or Btl transcription were obsenred in later stage 13 Doktlq homozygotes (results not

Defects in tracheal structure could be detected at stage 16. although visualization of the

completed tracheal system in Dakrlq homozvgotes was difficult because the MAbZA 12 antibody

staining was much weaker than in wild-type embryos (Figure BI). The dorsal trunk had an

abnormal wave-like structure. while other smaller branches (most obviousiy in the antero-dorsal

region) were stuntrd or abnormally-shaped.

Since it has been shown that the activation of marnmalian PKB is dependent on the

activity of phosphatidylinositol 3-kinase (PI-3K). 1 explored the possible conservation of

Drosophila PI-3K as a positive regulator of DAktl in the Trh signaling pathway. The tram-

heterozygous enhancer screen suggested that the Dp60 regulatory subunit. not the DpllO

catalytic subunit, of D P I J K is required for D.4ktl signaling. When Dakr14 heterozygous virgin 41

females were crossed to males carrying a deletion of a chromosomal region that includes the

gene that encodes the Dp 1 10 catalytic subunit of DPI-3K (Weinkove et al.. 1999). the lethality

seen in the ernbryos from this cross was only 2.4% (Figure 7A). The D ~ I lo4 deletion includes

the gene Hairless (H); however. the deletion is partially rescued by a H transgene ( P g [ H f i ,

resulting in a Dp I l O-specific mutation (Weinkove et al.. 1999). In contrast. when the Dakr l q

mutation is in irons to a chromosomal deletion mutation that rernoves the p60 regulatory subunit

of DPI-3K (Weinkove et al.. 1999). it induces 12.5% embryonic lethality and displays an

apoptotic cuticle phenotype (Figures 7A and D). These p60 mutant embryos that 1 examined

carry a deletion of a chromosomal region that contains three genes including the gene encoding

the p60 regulatory subunit of Drosophila PI-3K; however. these ernbryos also carry a transgenic

construct (Pg[R I O / ) that rescues the other two genes in the deletion. resulting in a p60-specific

deletion @604) (Weinkove et al.. 1999).

1 used confidence interval analysis to determine the significance of the difference in

embryonic lethality that was observed in the Dokr I V x Dp l I 0' or Dakt l q x ~ ~ 6 0 ' ' cross when

compared to the embryonic lethality that was obsrrved in each of the control crosses. Using this

statistical analysis. the lethality that was observed in the DaktIq x Dpl l v 4 cross was shown to be

not significantly different than the lethality that was observed in each of the control crosses:

however. the lethality observed in the Dakrlq x ~ ~ 6 0 " cross was shown to be significantly

different than the lethality that was obsrrved in each of the control crosses (see Table 3.

Materials and Methods).

Although the effect on stage 1 1 Trh expression in the homozygous mutant p 6 ~ 4 embryos

was similar to that seen in the Daktlq homozygotes (Figure 85), the defects in Bi1 transcription

and the subsequent effects on tracheal branching, migration and structure were much more

severe (Figures 8K and L). The antero-posterior branches (the dorsal trunk and the visceral 42

branch) were missing completely, and the dorsal-ventral branches (the dorsal branch and the

lateral trunk) were unbranched and stunted.

The UAS/Grl L 4 gene expression system is used in Drosophila to study the effects of

ectopic expression of specific genes dunng development. Genes fused to LT.4S (gpstream

activation pquence) elements are responsive to the GAL4 protein. The GIIL-I gene can be fused -

to. and placed under the control of. a specific promoter with a characterized expression pattem:

therefore. the U.4S gene will be expressed in the characteristic pattem of the promoter that is

driving G.4L-l expression. Thus. U4SiG.4 L4 transgenic tly lines can be used to express proteins

ectopically in the embryo.

Using embryos donated by A. S. Manoukian. 1 showed that over-expression of the

inhibitory phospholipid phosphatase. DPTEN. results in weak Trh activity (as indicated by Bi1

trancription) when compared to wild-type embryos (Figure SN). The dazcgrherlessGA L 4

expression construct (Giebel et al.. 1997) \vas used to drive ubiquitous stage 1 O expression of the

U4SDPTEiV transgene. Lastly. A. S. Manoukian donated DPTEI\"@~ GLC mutant embryos for

Btl in sitir hybridization. We found that these embryos not only have cxpanded Br1 transcription

around the normal tracheal pits but also that an anterior ectopic region of Br1 transcription could

be induced (Figure 80) . The loss of DPTEN results in ectopic but spatially-constricted Trh

activity in a manner that is similar to the effect seen with ubiquitous esogenous expression of

Trh (Wilk et al.. 1996) (see Figure 9.4). Thus. the genetic data suggested that DAktllDPI-3K

signaling positively regulates Trh transcriptional activity in the embryo.

3.3 The P element-Induced Pi627 Mutation

A non-embryonic lethal P element-induced mutation of Dukrl. P l 62 7. was discovered by

A. S. Manoukian using a Daktl nucleotide sequence homology search on the sequence database

from the Berkeley Drosophila Genome Project. I showed that this mutation did not complement 43

the Dakrlq mutation and could be rescued to adulthood with the Duktl transgene (results not

shown). Two hundred and twenty-four adults were scored in the complementation cross and al1

were balanced over Tm3. Sb. From the Daktl transgene rescue cross. 8 non-Sb rescued adults

were observed in the 1 19 flies that were scored. Furthemore. using an FRT P 162 7 mutant line, I

used GLC analysis to show that this mutation had a ce11 death phenotype (as assayed by cuticle

analysis) that was very similar to that of DaktlWLC embryos (results not shown).

In Pl627 mutants, the P element insertion in the Daktl gene contains a LacZ mini-gene

that. due to its insertion into the promoter region. can be expressed in a Daktl-specific pattern

and is detectable by anti-P ça1 antibody staining. This provided a novel way to examine the

zvgotic DaktI expression pattern in embryos since the previous study of Duktl expression had

used in situ hybridization (Franke et al.. 1994). An@ gal antibody staining using Pl627 mutant

embryos revealed a ubiquitous background expression pattern. highlighted by regions of strong

expression in the invaçinating put cells and in a pattern which coincides with Trh expression at

stage 10 in the tracheal cells surrounding the tracheal pits and in the antero-dorsal epithrlial cells

directly adjacent to the pits (Figure 8M).

3.4 Differential Effects of the ~ r h ~ " " and ~ r h " * ~ ~ " " Ectopically-Expressed Proteins

Ectopic expression studies using the transgenic Lr44SiG.4 L4 pattern-specific gene

BN17 expression system allowed me to study the differential effects of the wild-type Trh and

mutant ~ ~ ~ S e r 6 6 5 : ! ' 3 proteins in the embryo. As described previously (Wilk et al.. 1996).

ubiquitous over-expression of wild-type Trh at stage10 of embryonic development, using the

heat-shock inducible hsp70G.4L-l driver. resulted in spatially-resuicted ectopic domains of Trh

expression and Br1 transcription. 1 used the urm~1ciiiIo(arm~GAL4 line to drive ubiquitous

expression (Sanson et al.. 1996). starting at stage 10 and coincident with endogenous Trh

expression, of the wild-type L ~ I s T ~ ~ ~ " " protein with identical results (Figure 9A). However.

no ectopic Trh expression or Btl transcription was detected in identical experiments using

uASTrhk66j.4h . suggesting that the Trh Srr665t\la mutant is transcriptionally-inactive, since it is no

B.Vl7 longer a DAktl substrate (Figure 9B). Over-expression of wild-type Trh rescues Btl

transcription in Trh mutant embryos while over-expression of the Trh Ser665.41~ mutant does not

rescue Br1 transcription (Figures 9C and D). Of the 90 stage 10 embryos that were scored from

the uAsTrhSfl665:!h rescuc cross. 21 embryos (which is 23% of an exprcted 25%. assuming

normal chromosomal segregation in a cross between trh-'83 heterozygous parents) showed no

normal or ectopic Trh activity (as assayed by BtI transcription). Al1 of the 87 embryos from the

U A S T ~ ~ ~ " ' ~ rescue cross had Trh activity at both the normal and the ectopic sites.

Using hsp 70G.A L4 to over-express ~!-4s~rh~""~*"" at approximately stage 7. just p io r to

the expression of endogenous Trh. disrupted Btl transcription (Figure 9F). Thus. the

transcriptionally-inactive ~ r h ~ " ~ ~ ' " ' " mutant protein c m act as a dominant-negative inhibitor of

Trh activity.

The hypothesis that DAktl is required for Trh transcriptional activity was further

BN17 supported by the observation that ectopically-expressed Trh in a Doktlq homozygous

background. where the amount of DAktl protein is reduced and DAktl activity should be

limited. was unable to induce ectopic Br/ transcription in the majority (an estimated 80%) of the

Daktlq mutant embryos (Figure 9E). Of the 84 stage 10 embryos from this cross that were

scored for ectopic sites of Bi1 trancription. 17 embryos (20% of an expected 25%, assuming

normal chromosornal segregation in a cross between Daktlq heterozygous parents) were missing

the expected ectopic transcription sites. When armodilloGI1L-l or pairrd(prdjGrlL4 was used to

drive CO-expression of wild-type Trh with either DAktl or mammalian PKB in double UAS

transformant Drosophile Iines ( i v ; UA SDakt L'C'70; L ! . L s T ~ ~ ~ " * ~ and IV; USPKB; U ~ S T . ~ . ~ ~ ) , 45

Figure 9-Ectopic expression of the wild-type and mutant Trh proreins

Figure: Embryonic expression of wild-type and Ser665Ala mutant Trh proteins using the

UAS/GAL3 system.

amrGAL4-specific ectopic expression of wild-type Trh in a wild-type genetic background

(FigureaA), in a rrli mutant background (Figure 8C) and in a Daktlq background (Figure

8E) is shown. Figure 88 shows cinrrGAL1-driven ubiquitous expression of the Ser665Ala

mutant Trh in ii wild-type genetic background. Figure 8D shows expression of Ser665AIa

Trh in a rrli mutant background and Figure 8F shows ubiquitous expression of the mutant

Trh protein just pnor to the expression of endogenous Trh. Embryos were photogrciphed

at 20x magnificlition. For al1 embryos. anterior is to the left and dorsal is up.

only the normally-occurring sites of ectopic Bd transcription were observed (results not shown).

3.5 Both Trh Proteins Are Expressed Using the UASIGAL4 Expression System

The 5 ' end of each of the irh transgenes. L X S T ~ ~ ~ . " ' and ~i4~7'rh~'~""" . was Fused to a

small sequence encoding the FLXG@ protein. Thus. each ectopically -expressed protein was

' tagged' at its N-terminus with FLAG. The FLAG epitope from Sigma-Aldrich Co.. is a small

hydrophylic polypeptide consisting of 8 amino acids (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys).

Due to its small size and hydrophylic nature. the FLAG epitopc: is expected to be located on the

outer surface of the fusion protein and is not likely to alter the function. secretion or transport of

BN 17 the protein. To test the ability of the L7.-fS/G.-IL4 system to express both the Trh and the

Trh!kr66'hfil proteins in the ectopic expression experiments. Western blot analysis using the anti-

FLAG antibody against protrin rxtracts from 0-8 hour orm G A L A'u'.-f ~ ~ r h ~ . " ' - and

a r m ~ r l L - I / c ~ ~ s T ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ embryos was perfomrd (Figure lOA, Irnes 3 and 2, respectively).

The blot showed that both proteins were being ectopically-expressed using the LrAS/G,4L-I

expression system in the embryos. An ernbryonic extract from 0-8 hour wild-type embryos

showed that there is no endogenous FLAG epitope in Drosophikr at that stage (Figure 10A,

Lane 1). The size of the FLAG-tagged proteins was approximately l4OKda. which was in

agreement with the bacterially-expressed protein. as purified on an anti-FLAG column (Jing Jin

persona1 communication).

B.VIT In addition. pcrired (prd) G.4 L 41 UJSTrh and prdGJ L.// ~ ~ . 4 ~ ~ r h ~ ' ~ ~ ~ ~ . ' " embryos.

where GAL4 is expressed in altemating epithelial stnpes. were stained with anti-FLAG antibody

(Figures 10B and C). The results from these experirnents showed that both proteins were being

ectopically-expressed in the embryo with the UIISiG4L-I expression system.

3.6 DAktl Control of Trh Nuclear Translocation

Confocal fluorescence microscopy revealed that the exogenous FLAG-tagged wild-type

Trh protein localizes to the nucleus of al1 tracheal cells. including those of the ectopic tracheal

pits. as well as the adjacent dorsal rpithelial cells (Figure IOD). The ventral epidermal cells did

not show Trh nuclear translocation. A similar dorsally-restricted pattern of Trh activity (not Trh

nuclear localization) was observed in a previous study of Trh over-expression (Zelzer et al..

1997). The expression pattern of endogenous DAkt 1 suggested that DAkt l itself might be

required for Trh nuclear translocation in the dorsal epiderrnal cells: however. ectopic co-

expression of DAktl with Trh did not affect the spatially-restricted nuclear localization of the

exogenous Trh protein (results not s h o w ) . The mutant Trh Scr665:Ua protein is not able to

translocate to the nucleus in any cell in the embryo (Figure 10E). These results suggest that the

mechanism for DAktl regulation of Trh activity is through phosphorylation of Ser665 and that

this phosphorylation is almost certainly required for the nuclear translocation of the Trh protein.

Figure 1 O-Expression and cellular localization of ectopically-expressed Trh protein

Figure: Ectopic expression of wild-type and Ser665Ala mutant Trh proteins.

An anti-FLAG Western blot from 0-8 hour wild-type. arnlGAL4; U A S T ~ ~ ~ ~ ' " and

ann GA LI: U A S T ~ ~ ~ " ~ ~ ~ ' ~ ' " whole-embryo protein extracts is shown in Figure 1OA (lanes

1. 3 and 2, respectively). The protein marker on the right-hand side of the blot indicates

the molecular weight of the proteins in Kda. Anti-FLAG antibody staining of prdGM4:

U A S T ~ ~ ~ ~ . ~ " and prclGA LI; UAST~~Z~'""*~'" embryos is shown in Figures 1OB and C.

respectively. These embryos were photographed at 10x magnification. Con focal

microscopy was used to analyze the cellular localizntion of enogenous Trh around the

tracheal pits in anti-FLAG stained rinriGrlL4-l; U A S T ~ ~ Z ~ . ~ " (Figure 1 OD) and arnrGAL4; u ~ ~ ~ ~ / ~ S t r 6 6 C i \ l ( i (Figure IOE) embryos. Two adjacent tracheal pits are shown for each

figure. These embryos were scanned ai 63x rnagni fication. For all embryos anterior is to

the left and dorsal is up.

4. Discussion

4.1 Trh Implicated as a DAktl Signaling Pathway Component

The rrans-hcterozygous enhancer of lethality screen was used to identify mutations in

Drosophilu that interact with the Dakrlq larval-lethal mutation in a dose-dependent manner.

Interacting mutations induced lethality during embryogenesis while in rruns to Dakrlq. The P

element-induced allele of rrh and the chromosomal deletion of the p60 regulatory subunit of

Drosophila PI-3K were s h o w to have a genetic interaction with Dakf lY that resulted in

embryonic lethality. Less than 20 Drosophilu mutations were scored before identi@ing the

interaction between the trh and p60.' mutations with the DokrlY mutation. However. the rrh and

p604 mutations were not the only two mutations that induced significant lethality in rruns to

DnkrIq. Table 6 shows that at least two other P rlement-induced mutations (PI-lIO and P1661)

induced embryonic lethality at a level greater than that observed in wild-type embryos while in

rruns to Dakt14. This suggests that DAktl may be required for other processes during

development and therefore one or more components of additional signaling pathways could

interact with the Dakt Iq mutation. The ubiquitous expression of DAkt 1 during embryogenesis.

its essential role in ce11 survival and its universal requirement for cell size regulation seem to

support this idea. In the continuation of this screen. it will be interesting to see if the rate for

identiQing lethality-inducing mutations remains as high.

Since neither Daktlq nor P l 74- (rrh) are embryonic-lethal mutations. the lethality that

was induced in the DakrlVrrh rrans-heterozvgotes seems unexpected. Trh was showm to be

downstream of DAktl and has never been shown to affect ce11 survival; aside from tracheal

development. the only other documented requirement for Trh during embryogenesis is in the

development of the salivary glands (Isaac and Andrew. 1996; Wilk et al.. 1996). Trh is

expressed in the salivary gland primordia and is required for invagination of the tube-forming

duct cells of the salivary gland (Isaac and Andrew, 1996; Wilk et al.. 1996). Strangely, neither

of these Trh hnctions is consistent with an ability to induce lethality in Dakr l q hrterozygous

embryos. If Trh were an upstream positive regulator of D u t 1 kinase activity (as is expected of

DPI-3K). then some lethality might be expected in mutant rrrins-heterozygotes since the ce11

survival role for DAkt 1 has been previously documented (Staveley et al.. 1998). Perhaps the

lethality seen in these truns-heterozvgotrs is indicative of communication between the Trh

branch of the DAktl signaling pathway and the ce11 survival branch of the pathway.

Nonetheless. the utility of this simple screcn scems to have been validated by the genetic and

transgenic data that were used to characterize the positive regulation of Trh by DAkt 1 in vivo.

4.2 Genetic Analysis Revealed That DAktl Signaling Positively Regulates Trh Activity

Analysis of homozygous Daktlq mutant embryos revealed a weak cffect on Trh

expression in stage 1 1 embryos but not in earlier stage 10 embryos. This seemed to indicate that

the Duktlq mutation was not affecting the primary expression of Trh but was instead affecting

the secondary transcription-dependent auto-regulatory expression of Trh. Thus. it appeared that

the Trh regulatory feedback mechanism. whereby the very low-level primary Trh expression

m u t be amplified to higher levels by the activation of the transcription-dependent auto-

regulatory feedback loop. is dependent on DAktl activity. The hypothesis that DAktl may be

affecting Trh transcriptional activity was fùrther supported by the result that Bd transcription was

reduced in Dakrlq homozvgous embryos. The effect on Trh activity is weak due to the matemal

contribution of DAktl that partially rescues embryonic kinase requirements and subsequently

permits partial Trh activity in the DakflY homozygotes. Full Trh activity appears to require full

DAktl kinase activity and this kinase activity is obviously pemirbed in Dakrlq mutants. 53

I t was surprising that there was no effect on Trh expression in later stage 13 embryos

since the maternally-deposited DAkt 1 has presumably been gradually disappearing since the start

of embryogenesis. Since no effects on Trh expression were observed in later stages of

development, this suggests that either the Trh auto-regulatory pathway becomes independent of

DAktl kinase activity in later stages or that the phosphorylation of Trh at earlier stages is

somehow sufficient to maintain Trh auto-regulation throughout development. Hoivever. the lack

of an effect on Btl transcription in later stage embryos is not surprising since the maintenance of

Btl expression has been shown to be independent of Trh activity and is instead dependent on the

activity of another transcription factor. Drifter (Anderson et al.. 1996). Regardlrss. the sarly

effects on Trh activity do affect the final structure of the tracheal system. Most obviously. the

wave-like pattern of the main dorsal tmnk in DakrlY mutant embryos would seem ro indicate

directional branch migration defects that are due. in part. to the weak Bi1 transcription seen at

radier stages in these embryos. The strong dependence of tracheal branching on the Btl receptor

signaling mechanism would certainly indicate that any disruption of the pathway. even a slight

one. would result in an abnormal branching pattern.

Visualization of the tracheal system using the MAb2.412 antibody in D a k t l Y

homozygotes was difficult because expression of the hWb2A 12 epitope appears to be affected in

these mutant embryos. However, MAb2A 12 epitope expression was not affected in p604 mutant

embryos. Since the p60.' mutation obviously affects Trh activity. this suggests that the

MAbZA 12 epitope is a downstream target of DAkt 1 in a p60- and Trh-independent pathway.

Analysis of mutant embryos revealed that the regulatoq subunit of DPI-3K is an

important regulator of Trh activity. Although the effect of the p604 mutation on Trh expression

was similar to that seen in DaktlY embryos. it had a more severe effect on Btl transcription and

tracheal structure. The effect of the p604 mutation on Trh activity was presumably due to the 54

loss of DPI-3K-dependent DAktl activation. However, since the p604 mutant phenotype was

not rescued by ectopic over-expression of DAktl, the genetic epistasis has not been confirmed

for this pathway. Thus. it is not cenain that the effect seen in the p604 mutants is due to the loss

of D A M kinase activity. even though interaction of the Dnktlv and p60' mutations in the tram-

heterozygous enhancer of lethality screen indicated that these two genes are in the same

pathway. As described in Section 1. much evidence supports the hypothesis that mammalian PI-

3K is required For the activation of PKB and that the PKB signaling pathway is conserved in

Drosophila. In addition. DAkt 1 c m rescue defects in ey e development that are induced by over-

expression of dominant-negative DPI-3K (Scanga et al.. 1000). For these reasons it is not

unexpected that the loss of DPI-3K activity in p60' mutant embryos would lead to reduced

DAkt 1 activity and a subsequent reduction in Trh activity.

Sorne of the regions of Br1 transcription in p604 mutants were srnaller than wild-type or

were even missing. Considering the ce11 s i x and ceil survival functions of DAktl. the p604

phenotype could indicate a trachral ceIl size or ce11 death effect. However. in contrast to the

regions of Btl transcription in these embryos. the regions of Trh espression are not srnaller than

the regions of Trh expression in wild-type embryos. nor are they missing. Thus. the tracheal

cells in p60' mutant embryos are al1 present and appear to be normal in size. This would suggest

that in p 6 v 1 mutant embiyos the loss of Br/ transcription in some of the tracheal cells is due to a

loss of Trh activity. not due to a severe ce11 size or ce11 survival phenotype.

The p60'1 mutation had a much stronger effect on Trh activity than did the DaktlY

mutation. However. the loss of DAktl activity in the p604 mutant embryos did not result in a

severe ce11 death phenotvpe with severely-darnaged embryos that were difficult to analyze and

interpret (as in the Daktlq GLC embryos). In fact, the p6@f mutation does not induce embryonic

lethality and it has been suggested that the maternal contribution of p60 is responsible for 55

rescuing the embryonic development of p 6 ~ 4 homozygous embryos (Weinkove et al., 1999),

although gemline clone analysis has not been performed with these mutations to determine the

effects of complete loss of DPI-3K expression during embyogenesis. Thus. it appears that the

p604 mutation has a much stronger effect on DAktl activity but that there is enough matemally-

derived DPI-3K activity to permit DAktl ce11 survival signaling.

Mammalian PI-3K has been shown to be an intracellular mediator of multiple PKB-

activating growth factor signaling pathways. Growth factor receptors from the class of receptors

known as ~eceptor tyosinc kinases (RTKs). named because of their auto-phosphorylation at

tyrosine residues tollowing activation. have been shown to stimuiate P I 4 WPKB signaling.

Among these receptors are the mammalian FGF and EGF (Epidermal Growth Factor) receptors

(Burgering and Coffer. 1995; Yao and Cooper. 1995). The importance of Drosophila FGF

receptor (Btl) signaling dunng tracheation has already been discussed. In fact. the p6@f tracheal

structure phenotype. as indicated by MAb2.412 staining, is almost identical to the çffect seen on

tracheal structure in brl mutants (Klarnbt et al.. 1992). Drosophila EGF receptor signaling also

plays an important role in tracheal development. EGF receptor signaling is responsible for

determining antero-posterior specific tracheal branch fates. and disruption of this pathway results

in a tracheal structure phenotype that is also very similar to the p60" MAbîAlZ-specific

phenotype (Bier et al.. 1990: Wappner et al.. 1997). Mutations in the EGF receptor signaling

pathway result in a tracheal structure that completely lacks the antero-posterior specific branches

(the dorsal tmnk and the visceral branch). There are a number of ditrerent pathways in which the

p60 regulatory subunit of DPI-3K (and possibly DAktl) may be acting to affect tracheal

development; thus, the tracheal structure defects rnay not be solely due to defects in DAkt l n r h

signaling (Figure 1 1).

Figure 11-Receptor Tyrosine Kinase signaling in tracheal cells

Insulin FGF EGF

1 DAktl 1 ? 1 DAktl 1 ?

Branching Morphogenesis

Branch Fates

Btl (FGFR) - Expression

Figure: Diagrammatic representation of RTK signaling in tracheal cells. I have shown the requirement for DPI-3K and DAktl, signaling from an unidentified receptor, in Trh-dependent Bri transcription. The involvement of DPI-3K and DAkt 1 signaling in other tracheal ce11 RTK signaling path- ways (shown above) has been implicated by research done in mammlian systems studying PI3WPKB signaling.

The inhibition of DPI-3 WDAkt l/Trh activity by DPTEN was demonstrated by the

exogenous expression of a DPTEN transgene that inhibited Btl transcription. while the

endogenous function of this phosphatase as an inhibitor of DAktl signaling was shown using

GLC analysis with a DPTEN mutation. In the D P T E ~ V " ~ ~ ' GLC embryos. the area of Btl

transcription surrounding the tracheal pits appeared to be expanded when compared to that seen

in wild-type embryos. This phenotppe by itself may not have indicated that DPTEN was

involved in Trh signaling since the effect could have been due to a Ioss of the DPTEN-induced

inhibition of DPKWDAktl ce11 size regulation. The bigger tracheal cells would create the

appearance of an expanded region of Br1 transcription. This possibility was not explored and

cannot be mled out. Hoivever. an ectopic patch of Br/ transcription was induced in DPTEN GLC

ernbryos. This phenotype could not be due to ceIl s i x effects and shows that DPTEN can

specifically control Trh activity and BtI transcription. Thus. DPTEN is a component of the DPI-

X'DAktl/Trh/Btl signaling pathway. in addition to its function in the c d s i x regulatory

pathway. Trh activity can be directly controlled by DPTEN. which suggests that the endogenous

tracheal pits only appear bigger due to an increase in Btl transcribing cells surrounding the

tracheal pits and not due to an increase in tracheal ce11 size.

These data also suggest that in wild-type embryos. there is low-level Trh expression at

the ectopic site of Btl transcription and in an expanded ring surrounding the endogenous regions

of Trh expression. DPTEN must also be active in these regions to inhibit DPI-3K-induced

DAkt 1 activation; thus. without DAkt 1 activity. Trh cannot initiate the auto-regulatory feedback

loop. Without the amplification mechanism. Trh cannot reach sufficient Levels to activate Br1

transcription at the ectopic site or in the region surrounding the endogenous sites of Trh

expression. In addition. DPTEN must not be active at the endogenous sites of Btl transcription in

the wild-type embryo, in order to allow the activation of DPI-3K. DAktl and Trh. Thus, in the

absence of DPTEN, DPI-3K c m activate DAktl, which can then activate Trh.

1.3 PI 62 7 Expression Pattern

DAktl has been reported to be uniformly and ubiquitously expressed (Franke et al..

1994). 1 used P162l embryos to contirm that DAktl is expressed ubiquitously. and is also

strongly expressed (above the ubiquitous background staining) in the cells of the tracheal pits

and in the antero-dorsal epithelial crlls directly adjacent to the pits. Since the data suggests the

spatial and temporal CO-localization of DAkt l and Trh. it is consistent with a requirement for

DAktl in Trh signaling.

Furthemore. DAktl appears to be expressed in the anterior and posterior invaginating

eut cells. which suggrsts a rolc for DAktl in another morphogenetic tube-forming C

developmental pathway that could be similar to the tubulogenic Trh signaling pathway.

4.4 Transgenic Erperiments Demonstrate the Requirement of the Ser665 Residue for Trh

Activity

In vitro phosphorylation experiments performed by Jing Jin revealed that the Trh protein

was a target of m m a l i a n PKB and that the specific site for this phosphorylation was the serine

residue at position 665 within the PKB consensus site at residues 660-666. I showed that this

residue is required in vivo for Trh activity in the ectopic regions of Btl transcription that can be

induced with ubiquitous expression of a wild-type Trh transgene. In addition. the Ser665Ala

mutant Trh protein could not rescue Trh activity in rrh mutant embryos. Yet despite the in vitro

evidence that Trh is a target of PKB. this point mutation could have disrupted Trh activity by

some other mechanism. Prrhaps the structure of the Ser665Ala mutant pr~tein was disrupted.

which prevented the protein from activating transcription. However. the phenotype seen with

over-expression of the Ser665Ala mutant was mimicked by the over-expression of wild-type Trh 59

in a Dakt14 mutant background. suggesting that it is indeed DAktl activity that is required for

Trh activity. However. in the Pl627 expression experiment. there does not appear to be any

specific DAktl expression in the regions where the ectopic tracheal pits form. This would

suggest that there is some spatially-restricted DAktl activity at the sites where ectopic Br1

transcription can be induced which escapes DPTEN inhibition and is sufficient for Trh activation

in these regions whrn Trh is rctopically-expressed there.

In wild-type embryos. the endogenous levels of primary Trh expression and background

DAktI activity are too low to activate the Trh auto-regulatory feedback amplification loop at the

sites where ectopic Btl transcription can be induced. Without this amplification. Trh levels are

not sufficient to induce Br1 transcription. This may be due to the inability of these proieins to

tind and interact with each other in the cell. However. when Trh is ectopically-expressed at high

levels in these regions. the probability of an interaction between Trh and its activator. DAktl. is

increased. The weak background expression and activity of DAktl must be sufficient to activate

Trh in these ectopic regions. leading to subsequent Trh amplification and Br1 transcription.

Co-expression of Trh with either DAktl or PKB did not induce ectopic Bi1 transcription

outside of the regions where Btl transcription can be induced with the ectopic expression of Trh

alone. Since Tgo is ubiquitously-expressed. this suggests that there is another element(s) that is

required for Trh activity. This element must be spatially-restricted to both the endogenous sites

of Btl transcription and the ectopic sites of Btl transcription that forrn when Trh is over-

expressed. One possible candidate for this elernent(s) is a DPI-3 WDAktl activation signal. It is

very likely that the limiting factor in Trh activation is the localized activation of DAktl that is

restricted to the enodogenous sites of Btl transcription and. at a lower level. to the sites where Btl

transcription c m be induced when Trh is over-expressed.

The heat-shock inducible hspGI1L-l promoter was used to test the possible dominant-

negative effect of the Ser665.41a mutant Trh protein, because it c m be used to drive expression

at almost any stage dunng embryogenesis. The arrnG.4l-l promoter drives expression starting at

around stage 10. which is coincident with rndogenous Trh expression. Under the armG.4 L4

promoter. the erogenous Ser665Ala Trh protein may not have the opponunity to express enough

protein early enough to interfere with endogenous Trh signaling. However. when the

hsp7OGILL-l promoter was used to express the mutant Trh Srr665Ala protein at stage 7 and thus

allow a sufficirnt concentration of mutant protrin to accumulate prior to endogenous Trh

espression. endogenous Btl transcription was disrupted. Thus. the transcriptionally-inactive

TrhS~r665t\ll can act as a dominant-negative inhibitor of Trh activity. possibly by partially out-

competing the endogenous protrin for its binding panner. Tgo. The inhibitory effects of a

dominant-negative tnnscriptionally-inactive Trh protein has been previously observed (Wilk et

al.. 1996). In this previous study. a mutant Trh protein was created that cmied a delrtion in the

putative 'transcription activation' domain near the C-terminus. Over-expression of this mutant

Trh protein, using hsGAL4. dismpted tracheal cell invagination. as indicated by tracheai lumen-

specific antibody staining (Wilk et al.. 1996).

4.5 Mechanism for DAktl Control of Trh Activity

Cellular localization of the exogenously-expressed Trh proteins using anti-FLAG

confocal fluorescent microscopy suggested that phosphorylation of the Su665 residue is

required for nuclear localization of Trh protein. Thus. D A M regulates the ability of Trh to

traverse the nuclear pore cornplex and reach its target DNA within the nucleus. However. the

data does not address the regulation of Trh nuclear localization by DAktl. D A M

phosphorylation of Trh may be required for Trh::Tgo heterodirnerization (which has been s h o w

to be essential for nuclear translocation) (Sonnenfeld et al.. 1997). Alternatively. DAkt 1 may be

required for nuclear translocation after Trh::Tgo heterodimerization (Figure 12).

4.6 The Study of Oncogenes in Drosopltila

The study of an oncogene such as PKB can lead to a better understanding ot' the process

of oncogenrsis and the development of cancer. Transient expression studies with marnmalian

PKB in ce11 culture have uncovered a variety of functions for PKB. ranging from glucose

metabolism to protein synthesis to apoptosis. However. in vivo mutational analysis during

development using the homologues of PKB from Drosophila melanoguster (Scanga et al.. 2000:

S tavrley et al.. 1 998) or from the worm Coenorhabditis elegans (Ogg and Ruvkun. 1 998: Paradis

et al.. 1999: Paradis and Ruvkun. 1998) represent a more biologically-relevant and informative

way to study PKB signaling. The study of DAkt l/Trh signaling in Drosophilu may be used to

fiu-ther delineate the possible role for PKB in the oncogenic tubulogenesis-dependent process of

tumor vascularization.

Recently. it has bern suggested that PKB is involved in the mammalian tubulogenic

processes of vasculogenesis (formation of new vessels) and angioçenesis (formation/outgrowth

of vessels from existing ones) through the regulation of the mammalian transcription factor

Hypoxia-Inducible Factor 1 -a (HIF 1 -a) which has sequence homdogy to the Drosophilu rrh

gene (Isaac and Andrew. 1996: Mruurr et al.. 1997: Wilk et al.. 1996). Mammalian PI-3K was

s h o w to play a role in the hypoxia-dependent activation of HIFI-u. thus implicating the

involvement of PKB as an upstream activator of this Trh-like protein (Manire et al.. 1997).

Furthemore. mamrnalian PTEN has been shown to negatively regulate HIF- 1 c( transcriptional

activity through a mechanism involving the deregulation of PKB activity (Zundel et al.. 1000).

Figure 12-Phosphorylation of Trh by DAkt 1

non Receptor Activation

Nuclear Transiocation

Figure: Diagram depicting the two possible steps for DAkt 1 phosphoryl ation of Trh. It remains to be discovered exactly how DAkt l regulates Trh trans- location. Phospory lation may be requi red for Tgo heterodimerization or it could be required for the nuclear translocation of the heterodimer afier the two proteins have already bound to each other.

In response to hypoxic conditions. HIFI-a forms a heterodimer with the constitutively-

expressed HIF 1 -p/Amt protein. which is the mammalian homologue of the Drosophila Tgo

protein (Ema et al.. 1997; Ryan et al., 1998). and forms the transcriptional activator HIF-1

(Wang et al.. 1995: Wang and Semenza. 1995). Subsequent to its activation. HIF-1 is required

for the expression of rnany hypoxia-inducible genes. including vasculrrr rnciothelial groivth

factor ( FEGR.

VEGF is important for the tubulogenic processes of vasculogenesis. angiogenesis. ceIl

migration and the maintenance of existing blood vessels (Brown et al.. 1993: Brown et al.. 1992).

These processes are required during normal rnammalian development and are also required for

the 'angiogenic switch' in the hypoxic interior of tumors. Tumor growth is limited by the ability

of the vascular system to deliver sufficient osygen and metabolites to the growing cells: thus. a

tiimor's ability to develop new blood vessels as it grows (the switch to angiogenesis) is an

important step in determining the size of a tumor. Interestingly. VEGF has been shown to

activate the rnammalian PI-3 WPKB signalinç pathway : this activation promotes ce11 sunrival in

the endothelial cells of blood vessels (Fujio and Walsh. 1999: Gerber et al.. 1998). This anti-

apoptotic function is important for maintaining vesse1 integrity during the ce11 proliferation-

dependent events of neovascularization (during normal development or during tumor

angiogenesis). since ce11 death would be counter productive during this process (Dimmeler and

Zeiher. 2000).

Other evidence that implicates the involvement of rnamrnalian PKB in tubulogenic

processes includes the fact that the angiopoietin-1 (Anel) signaling ligand activates PKB in a PI-

3K-dependent manner and promotes rndothelial ce11 survival (Kim et al.. 1000). Angl signaling

is essential for embryonic blood vesse1 formation and integrity (Davis et al.. 1996). Lastly. PKB

has been shown to phosphorylate the endothelial nitric oxide synthase (eNOS). resulting in

constitutive activation of the enzyme (Dirnrneler et al.. 1999; Fulton et al., 1999). Nitric oxide is

essential for endothelial ce11 survival, ce11 migration and vascularization during embryonic

development (Dimmeler and Zeiher, 2000). PKB-dependent Angl and eNOS signaling is an

important component of normal mammalian tubulogenesis and may also be important during the

'angiogenic switch' in tumors (Dimmeler and Zeiher. 2000).

1.7 Tubulogenesis in Drosophila and Mammals

In both insects and mamrnals, the genesis of many essential organs is dependent upon the

processes of'tubu~ogenesis and branch formation for the development of multicellular. branched

cpitheiial tubules. The Drosophila ,eut. tracheal system. and salivary ducts are as dependent on

tube formation as are the mammalian lungs. kidneys and vascular system. The mechanism for

the development of these complex branching patterns in the tracheal system and in the

mammalian lung has been partially conserved between these diverse organisms. Tubulogenesis

in both organisms relies on ce11 shape changes. ce11 migration. interactive signaling between the

epithelium and the surrounding (mesenchymal/mesodermal) tissue and ce11 proliferation. (Note:

In contrast to each other. the mitotic events during Drosophila tracheal development only occur

prior to tracheal development whereas ce11 proliferation occurs throughout mammalian lung

development.) The use of similar signaling motifs in ordrr to accomplish similar tubulogenic

processes makes the study of Drosophila tracheal development useful in understanding

mammalian lung organogenesis (Hogan and Yingling, 1998: Metzger and Krasnow. 1999:

Warburton et al., 2000).

For example, Drosophiln tracheal development is dependent on: Btl/Bnl FGF receptor

signaling for the general branch migration mechanism (Klambt et al.. 1992; Lee et al.. 1996;

Sutherland et al., 1996). Dpp receptor signaling for defining the temporo-spatial expression of 65

the initial tracheal determinants and then for dorsal/ventral branch fate determination (Vincent et

al.. 1997; Wappner et al.. 1997) and lastly, EGF (epidermal growth factor) receptor signaling for

antero-posterior branch fate determination (Bier et al., 1990: Wappner et al.. 1997).

Similarly. mammalian h g oqanogenesis is dependent on FGF receptor signaling for

branch formation when the developing lung bud bifurcates to form the right and left bronchi

which then undergo tùrther branching events to form the branchioles and the alveolar ducts and

sacs (the main sites for gas exchange within the lung) (Bellusci et al.. 1997: Cardoso et al., 1997:

Peters et al.. 1994: Peters et al.. 1992). The homologues of Dpp. the TGF-B (transforming

erowth factor-6) farnily of secreted proteins. are essential for lung development: however. the C

different family members can ofien play contradictory roles. For example. TGF-6 receptor II

negatively regulates lung development while TGF-P receptor 1 positively instmcts the formation

of early branch points (Warburton et al.. 3000: Zhao et al.. 1996: Zhao et al.. 1998). Another

TGF-P farnily protein. BMP4. is thought to be required for the differentiation of proximo-distal

(or dorsal-ventral) epitheliurn in the primordial lung as it initially branches away from the ventral

foregut (Bellusci et al.. 1996). EGF receptor signaling appears to be required for late-stage

alveolization and differentiation of late-stage alveoli-associated epithelial ce11 types (Mieninen et

al., 1997; Seth et al.. 1993; Warburton et al., 1992).

Thus, the conservation of signaling motifs between mammals and Drosophila suggests

that studying tubulogenesis in insects can be informative for the study of mammalian

oqanogenesis.

4.8 Future Directions

One future project would be to determine the mechanism by which DAktl regulates Trh

nuclear translocation. In the case of the FKHRLl transcription factor in mammals. the

phosphorylation of FKHRL1 by PKB targets the protein for binding by 14-3-3 proteins (Brunet

et al., 1999). 14-3-3 proteins are a family of regulatory scaffold-associated molecules that are

able to bind their protein targets at a conserved sequence motif that contains a phosphorylated

serine or threonine residue (Fu et ai.. 7000). Following PKB phosphorylation. FKHRLI is

bound by the 14-3-3 proteins and retained within the cytoplasm where it cannot initiate

transcription of its pro-apoptotic target genes. In addition. PKB phosphorylation of the death

agonist. BAD. is thought to lead to its cytoplasmic retention through binding by 14-34 proteins.

thus removing it from its site of pro-apoptotic action at the mitochondria (Zha et al.. 1996).

However. Trh does not contain a 14-3-3 bindinç site consensus motif (A. Manoukian. persona1

communication). Furthemore. the DAkt 1 -dependent mechanism for controlling Trh activity by

regulating its cellular localization is permissive for Trh activity. whereas the previously-

mentioned PKB/I-l-3-3-dependent mechanism for controlling FKHRL l and BAD activity by

regulating their cellular localization. is restrictive. This suggests a novel mechanisrn for

controlling cellular localization of Trh that could simply be the sontrol of Trh::Tgo

heterodimerization.

The question of whether DAktl phosphorylates Trh before or after Trh::Tgo

heterodimerization remains to be answered. Perhaps CO-imrnunoprecipitation of PKB

phosphorylated wild-type and Trh Ser665Ala proteins with the Tgo protein might answer this

question. If Tgo CO-precipitates with the phosphorylated wild-type Trh but is unable to do so

with the non-phosphorylated Trh Scr665AIa protein. then it might be concluded that Trh::Tgo

heterodimerization is dependent on DAkt 1 phosphorylation.

The involvement of the Trh protein in the tubulogenic process of salivary gland

formation warrants an examination of salivary gland development in Daktlq mutants (Isaac and

Andrew. 1996; Wilk et al.. 1996). Another suggestion that DAkt 1 is required in the salivary 67

glands is the fact that marnrnalian PKB phosphorylates FKHRL 1, as well as recent evidence that

its Drosophila homologue, Forkhead (FKH), is required for ce11 shape changes during formation

of the salivary gland tubes (Myat and Andrew, 2000). In Fkh mutants, the salivary gland

precursor cells do not invaginate and the secretory tubes do not form. This effect is similar to

that seen for tracheal and salivary gland development in rrh mutants. suggesting that FKH could

also be a target of DAkt 1 during Drosophila ernbryogenesis.

Finally, it is obvious that the DubIq rrans-heterozygous enhancer of embryonic lethality

screen is incomplete. 1 have tested twenty of an estimated 2000 P-element induced mutant

Drosophila lines from the Bloomington Stock Center. The continuation of this simple screen

could prove to be extremely interesting and useful in Further delineating the role of DAktI

signaling during Drosophila development.

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