The Jing zinc finger protein: its transcriptional regulation by The Jing zinc finger protein: its transcriptional regulation by bHLH-PAS, POU, and ETS-domain transcription factors and its bHLH-PAS, POU, and ETS-domain transcription factors and its role in tracheal cell migration during role in tracheal cell migration during Drosophila Drosophila embryogenesisembryogenesis

Presented by Tatiana MorozovaPresented by Tatiana Morozova

April, 2010April, 2010

Department of Cellular and Molecular Department of Cellular and Molecular MedicineMedicine

Outline Outline 1. Introduction1. Introduction a. Development of a. Development of DrosophilaDrosophila tracheal system tracheal system b. Combinatorial transcriptional regulation in b. Combinatorial transcriptional regulation in DrosophilaDrosophila trachea trachea c. c. jingjing genomic structure and embryonic expression pattern genomic structure and embryonic expression pattern d. functions of d. functions of jingjing2. Hypothesis and Objectives 2. Hypothesis and Objectives 3. Results3. Results a. Generation of the 1.5, 1.3 and 2.8kb a. Generation of the 1.5, 1.3 and 2.8kb lacZlacZ reporter constructs reporter constructs b. Expression pattern of the 1.5kb b. Expression pattern of the 1.5kb lacZlacZ reporter construct reporter construct c. Expression pattern of the 2.8kb c. Expression pattern of the 2.8kb lacZlacZ reporter construct reporter construct d. Expression pattern of the 1.3kb d. Expression pattern of the 1.3kb lacZlacZ reporter construct reporter construct e. Transcriptional regulation of e. Transcriptional regulation of jingjing expression. expression. jingjing responsiveness to responsiveness to

RTK signaling RTK signaling f. The role of f. The role of jingjing during tracheal cell migration during tracheal cell migration 4. Conclusions4. Conclusions

IntroductionIntroduction

Model system – trachea of Model system – trachea of Drosophila melanogasterDrosophila melanogaster

A

Adapted from Beitel GJ, Krasnow MA, Development, 2000

Tracheal development and the roles of EGFR (DER) Tracheal development and the roles of EGFR (DER) and FGFR (and FGFR (btlbtl))

Stage 10: Placode. The last mitotic cell division.

invagination

Tracheal pit (80 cells)

EGFR (DER)

Stage 11-15: BranchingDirected cell migration,

primary branches.

Secondary branches

Terminal branches

FGFR (btl)

Relationship between Relationship between bnlbnl and and btlbtl expression during expression during

tracheal formationtracheal formation

pit

Bnl Btl

1º branching

Stage 12Stage 11 Stage 13/14

GBDTpDTa

DB

VB

LTa LTp

A. B. C. D.

• Bnl guides tracheal tip cells expressing btl• Btl activates MAPK at the tip of primary branches in cells destined for migration• Bnl is expressed in the cells surrounding placode at the 5 points where future primary branching will occur

Adapted from Ohshiro et al., 2002

Pit

DTp

DBDTa

LTa

VB

LTp

GB

Trh staining

RTK signaling and its regulation in tracheaRTK signaling and its regulation in tracheaEx. Transcriptional regulation of the Ex. Transcriptional regulation of the breathlessbreathless gene gene

Bnl

Btl

Ras/MAPK

Pnt Aop

ETS CME btl

Trh/Tgo

Vvl (Dfr) Sal

Spry

Positive feedback

Adapted from Ohshiro et al., 2002

jingjing genomic structure and embryonic genomic structure and embryonic expression patternexpression pattern

Adapted from Sedaghat et al., 2002

A

B DC

E F G

functions of functions of jingjing

jingjing is required in the CNS midline and trachea for patterning of CNS axons and is required in the CNS midline and trachea for patterning of CNS axons and tracheal tubulestracheal tubules

Loss-of-function mutations of Loss-of-function mutations of jingjing cause apoptosis in CNS midline and tracheal cause apoptosis in CNS midline and tracheal precursorsprecursors

midline repulsive mechanisms are perturbed in midline repulsive mechanisms are perturbed in jingjing mutant embryos mutant embryos

jingjing is required for midline glial survival is required for midline glial survival

jingjing is required for proper differentiation of midline glial and neuronal populations is required for proper differentiation of midline glial and neuronal populations

jingjing is required for both EGFR and FGFR-dependent MAPK activity is required for both EGFR and FGFR-dependent MAPK activity

jingjing is required for border cell migration in is required for border cell migration in Drosophila Drosophila ovariesovaries

jingjing is required for wing and leg development in is required for wing and leg development in DrosophilaDrosophila

Hypothesis

jing is responsive to tyrosine kinase signaling jing expression is regulated by Trh::Tgo, Drifter and Pointed in vivo jing is required for maximal levels of FGFR (btl) expression in the embryonic trachea

Objectives

1. To show that jing is responsive to tyrosine kinase signaling

2. To investigate transcriptional regulation of jing in trachea by Trh::Tgo, Pou-domain transcription factor Drifter, and ETS-domain transcription factor Pointed

3. To show that jing is required for maximal levels of btl expression in the embryonic trachea

ResultsResults

Generation of the Generation of the jing1.5-lacZjing1.5-lacZ, , jing1.3-lacZjing1.3-lacZ and and jing2.8-lacZ jing2.8-lacZ

reportersreporters

jing1.5-lacZjing1.5-lacZ, , jing1.3-lacZjing1.3-lacZ and and jing2.8-lacZ jing2.8-lacZ reportersreporters1.

5kb

2.8k

bla

cZ c

onst

ruct

la

cZ c

onst

ruct

Adapted from Sonnenfeld et al., 2010, submitted

lacZ

con

stru

ct

1.3k

bDN

A la

dd

er

DN

A la

dd

er

Embryonic expression pattern of the Embryonic expression pattern of the jing1.5-lacZjing1.5-lacZ reporter construct reporter construct

jing1.5-lacZ expression in the trachea sa

gitt

al

stag

e 14

sagi

ttal

st

age

11

B’’

B

B’’

sagi

ttal

st

age

11Db

Dt

Lt

P[jing1.5-lacZ]

A’’’

Dt

Db

merge

jing1.5-lacZ jing1.5-lacZ expression in the fusion cells (DB, expression in the fusion cells (DB, DT, LT)DT, LT)

sagi

ttal

st

14

dor

sal s

t 13

dor

sal s

t 14

red-fusion cells, blue-tip cellsAdapted from Hayashi et al., 1999

A’ A’’

B B’

C’’C C’ C’’

A

B’’

jing1.5-lacZjing1.5-lacZ expression in the midline and expression in the midline and neuronal bodiesneuronal bodies

ven

tra

l st

14

ven

tra

l st

14

Embryonic expression pattern of Embryonic expression pattern of the the jing2.8-lacZjing2.8-lacZ reporter construct reporter construct

jing2.8-lacZjing2.8-lacZ expression pattern in trachea and expression pattern in trachea and midlinemidline

sagi

ttal

st 1

2sa

gitt

al

st 1

5sa

gitt

al

st 1

5ve

ntr

alst

10 D D’ D’’

D D’ D’’

DB

DT

DTa DTp

Vb Tc

B’’DB

Dt

jing2.8-lacZjing2.8-lacZ expression in the fusion cells (DT, expression in the fusion cells (DT, DB, LT)DB, LT)

sagi

ttal

st

14d

orsa

l st

16sa

gitt

al s

t 12

red-fusion cells, blue-tip cellsAdapted from Hayashi et al., 1999

DB

DT

LTa

jing2.8-lacZjing2.8-lacZ expression in the dorsal branch fusion expression in the dorsal branch fusion cells, posterior spiracles, and segmental ectodermal cells, posterior spiracles, and segmental ectodermal

stripesstripes

dor

sal

st 1

5sa

gitta

lst

10

B

sagi

ttal

st 1

0

P[jing2.8-lacZ] anti-En merge

C C’ C’’

Embryonic expression pattern of the Embryonic expression pattern of the jing1.3-lacZjing1.3-lacZ reporter construct reporter construct

jing1.3-lacZ is expressed in segmental ectodermal stripes and in En-positive midline

neurons

B B’ B’’

ven

tra

l st

13

ven

tra

l st

14

jing1.3-lacZ is not expressed in the trachea s

agit

tal s

t 12

sag

itta

l st

15s

agit

tal s

t 15

Expression patterns of Expression patterns of jing1.5-lacZjing1.5-lacZ, , jing1.3-jing1.3-lacZlacZ, and , and jing2.8-lacZjing2.8-lacZ reporters reporters

jing1.5-lacZ jing1.3-lacZ

jing2.8-lacZ

fusion cells

midlineneuronal bodies

glia segmental stripes

midline

non-fusion tracheal cells

Transcriptional regulation of Transcriptional regulation of jingjing expression expression

Regulation of Regulation of jing1.5-lacZ jing1.5-lacZ expression by expression by Single-minded bHLH-PAS transcription factorSingle-minded bHLH-PAS transcription factor

simH9 – null allele

ven

tral

st

13

+, jing1.5-lacZ

ml

simH9/ simH9, jing1.5-lacZ

mlLE LE

A B

trhtrh, , dfrdfr and and pntpnt are required for endogenous are required for endogenous jingjing mRNA expressionmRNA expression

sagi

ttal

st

13sa

gitt

al

st 1

3

C D

w1118

pnt039/pnt039

embryos hybridized with Digoxigenin-labelled jing anti-sense riboprobes and stained with anti-Dig

Stable line 1: [Stable line 1: [jing1.5-lacZ/jing1.5-lacZjing1.5-lacZ/jing1.5-lacZ];[];[trhtrh22/trh/trh22]]

Trh and Tgo are required for jing1.5-lacZ expression in vivod

orsa

lsa

gitt

al

0

20

40

60

80

100

120

jing1.5-lacZ jing1.5-lacZ/trh2

DB weak

DT weak

LT weak

jing1.5-lacZ jing1.5-lacZ/trh2

B B’ B’’

Control jing1.5-lacZ

Db 1.5jing-lacZ n=123 metameresDb trh2 n=78Dt trh2 n=87

trh2 – mild hypomorph

% of the segments showing weak lacZ expression

Ectopic activation of the Ectopic activation of the jing1.5-lacZjing1.5-lacZ reporter by reporter by trhtrh and and dfr/vvldfr/vvl

btl

in

sit

uve

ntr

al

an

ti-β

-gal

ven

tral

st

12

A B C

controlprd-Gal4; jing1.5-lacZ

D E F

prd-Gal4; UAS-vvl,trhprd-Gal4; UAS-trh control prd-Gal4; +/+

prd-Gal4; UAS-vvl,trh; jing1.5-lacZ

prd-Gal4; UAS-trh; jing1.5-lacZ

Integrity of the CMEs is required for the regulation Integrity of the CMEs is required for the regulation of of jingjing expression by Trh::Tgo expression by Trh::Tgo in vivoin vivo and and in vitro in vitro

(previous results)(previous results)

C

sag

itta

l ve

ntr

al

st 1

5sa

git

tal

ven

tral

st 1

5

Dfr Dfr is required for activation of theis required for activation of the jing1.5-lacZ jing1.5-lacZ reporterreporter in in vivovivo

Stable line 2: [jing1.5-lacZ/jing1.5-lacZ];[DfrE082/DfrE082]

0

20

40

60

80

100

120

jing1.5-lacZ jing1.5-lacZ/Dfr

DB weak

DT weak

LT weak

jing1.5-lacZ jing1.5-lacZ/dfrE082

dor

sal

dor

sal

Dt DfrE082 n=56 metameresDb DfrE082 n=90

dfrE082 – strong hypomorph

dml

% of the segments showing weak lacZ expression

control jing1.5-lacZ

Stable line 3:Stable line 3: [[jing1.5-lacZ/jing1.5-lacZjing1.5-lacZ/jing1.5-lacZ];[];[pntpntE039E039//pntpntE039E039]]

jing1.5-lacZ activation in tracheal fusion cells requires the Pointed ETS transcription factor

dor

sal

dor

sal

0

10

20

30

40

50

60

70

80

90

100

jing1.5-lacZ jing1.5-lacZ/pnt

DB weak

DT weak

LT weak

jing1.5-lacZ jing1.5-lacZ/pntE039

B’B A’’

control jing1.5-lacZ

Db pntE039 n=163Dt pntE039 n=120

pntE039 – hypomorph(Klambt C)

% of the segments showing weak lacZ expression

ATGGATATTTATGAATGGATATTTATGCTTCGCGTGCCCGCTCGTAGAAACAGAAACGTCAGTGTGCGCGCGTGTGTTTGTGTGGATAATTAAAGCAGCAACGATTTCGAGTACGAAATGCTTTAAGACCGAGATTAAAGTTGAAGGTCGGTCGTTGTTGTTGTTGCGTTCGTCTGTGTTTGTGTGTGCATGTGTGTGGAA

AAGCGGACAGGAAAACGGATCGGAATCGGATCGCCTGCCTGGGAACACGCATAGCGTTAGCGTTGAACGTTAGTATACCGACGAAGGCTGAAGTCGTTAAGCCAAAGAATACGAATACGCCAGCAAAATAAAAGTAATCGTGCATTGCTCCCTCTCTGCTGCTGCGCATGTTTCTCTGCCGCCTCGCTGCTGCAGCGCT

GCCTCGGCCTGCATTTTTAGTTTCGCCCGACTATTACAGCATGCTTTACATTCCGTTTACATCACGTGTTAAATAACTGCTGCCTGTCACTGTTCGCTGTTCGCGACCGTGTGTGTGTAATTGTGTGCAATTGTTGTTGTTTGCCCAAGCAAATATGCAATGATTTTTGCGCCACCCCCAACCCCCATGCCATCTCTCCCCCTG

CAACCAGTGCCTTCTTCATTCATGCCTTGCGTGTATGTGTGTGTGTGTTGGATACACACCCCCACATCCGACCCACGCACACAAACACACGACAAAGGCAACTTCTGGCGGATGAGGAAGCCGCTTTCGGTGTGTGCGTGTGTATCTAATGGGATTTTCTAATAAAATTTAAATTGTTGCCGCTTTTGCCGTGCGGCGTTC

GTTCTATGAATTTAATATGACCCCTGTGTGTGTGAATAGGGGCCATAAAGCTTTGCTTATGGCTGGAAAATTAAATAGAGTGCATAATTTAAGCATCAACTTTGCGTTGAATGCATAATAAACGGCTGCAATTGAAAGGCGCCGTCTACCAAAGGACTACTGCATAAATATCGGATAACTTATACTCGGGCTCAGTGCCA

Pnt AOP/YanCCAGATTCACAATTTGCAACGGACGAAGGGAATGTAAGTCCCTGGAAATGGCCGTCGGCAAACAGAAAGTGTCTGATTATGTCGTTCAAAATGTTAGCT

ATATTAACTGCCAGTCAACGATACAATGTGTAGTTTAAAGCTGTCACGATTTTCGCAGTGCGTCTGTCAATTAATGAGCTTTTCAAATATATAAATATTAGTTCCCCGAAAAAATTTCAACAGTAAGGGATGCATACGGGTGATTTAAAATTACTTAAAAGAAATCATTCTGCTTAAATTTATATGCTTTAAACCAACATT

ACGTGATTTCAAATTTAAAAAATAATAGTGAATACCACTTTACGATAGCAGAGCTCTCAATTAAACCCTACTGTTTTAAAACAAGTGAGTGTGTACACAC

ATATACTGATGGTATTGATGTTTGTTTATAAAACGTACTCCATATTAAACATTACTTGACTACGTGTCCCTCAATTTGTTGTTAGAAATTAAACGAAATTTGAGTCCAACAGATTTCTGTGTCATATTTGAGTATGAATCCGCATCTGTAAAAA

CME1

CME2

CME3

CMEs

1(2)01094

A

B

mut5

Jing regulatory region Sedaghat Y. et al., 2002

Generation of the 1.5jingΔPnt-lacZ reporter

1.5j

ingΔ

Pn

t-la

cZ

Adapted from Sedaghat et al., 2002

DN

A la

dd

er

Activation of Activation of jing1.5-lacZjing1.5-lacZ reporter by ETS-domain reporter by ETS-domain transcription factor Pnt is dependent on the integrity of transcription factor Pnt is dependent on the integrity of

consensus ETS DNA binding sitesconsensus ETS DNA binding sites

jing1.5ΔPnt-lacZ jing1.5ΔPnt-lacZ expression patternexpression pattern

0

5

10

15

20

25

30

35

40

45

1.5jing lacZ deletion pnt

DB weak

DT weak

LT weak

jing1.5-lacZ jing1.5ΔPnt-lacZ

% of the segments showing weak lacZ expression

sagi

ttal

st

15sa

gitt

al s

t 15

dor

sal s

t 13

dor

sal s

t 16

A A’ A’’

B’’B’B

C C’ C’’

A

P[jing1.5ΔPnt-lacZ] anti-Tgo merge

B B’ B’’

A A

jing1.5ΔPnt-lacZ anti-Tgo merge

jing1.5-lacZ anti-Tgo merge

dml

CNS

fc

dorsal stage 13

dor

sal s

t 13

fcCNS

jingjing responsiveness to RTK responsiveness to RTK signalingsignaling

jingjing is responsive to EGFR signaling in midline glia is responsive to EGFR signaling in midline glia

ven

tral

st

13

control

Stable line 4: [jing1.5-lacZ/jing1.5-lacZ];

[rho7M/rho7M]

Stable line 5: [jing1.5-lacZ/jing1.5-lacZ];[bnl1/b

nl1]

control 1.5jing-lacZ

sagi

ttal

sagi

ttal

jingjing is responsive to EGFR and FGFR signaling in is responsive to EGFR and FGFR signaling in tracheatrachea

E

F

dor

sal

+ 0

10

20

30

40

50

60

jing1.5-lacZ jing1.5-lacZ/rho

DB weak

DT weak

LT weak

% of the segments showing weak lacZ expression

jing1.5-lacZ jing1.5-lacZ/rho

Db

Dt

rho7M – null allele

The role of The role of jingjing during tracheal during tracheal cell migrationcell migration

jing jing mutant embryos show defects in tracheal cell mutant embryos show defects in tracheal cell migrationmigration

jing function is required for proper cell migration during

tubulogenesis

+

sagi

ttal

sagi

ttal

dor

sal

sagi

ttal

sagi

ttal

jing1.5-lacZ/jing3 mergeanti-Tgo

F GEjing3

D D’ D’’

btlGal/UASjingRNAi btlGal/UASjingRNAiw1118

H I

w1118 jing3

jing3 –strong hypomorph

jingjing is required autonomously for maximal is required autonomously for maximal btlbtl expression in the tracheaexpression in the trachea

sagi

ttal

dor

sal

ven

tral

sagi

ttal

w1118

A B

btl-Gal4;UAS-jingRNAi arm-Gal4;UAS-jingRNAi

C D

arm-Gal4;UAS-jingΔ5N

w1118

E

jing3/jing3

F

w1118 btl-Gal4;UAS-jingRNAi

G H

w1118

sagi

ttal

I J

w1118 btl-Gal4;UAS-jingRNAibtl-Gal4;UAS-jing RNAi

K L

jing3 –strong hypomorph

Conclusions

jingjing CNS midline elements drive expression in tracheal fusion cells CNS midline elements drive expression in tracheal fusion cells

jingjing 2.8kb 5’regulatory region contains additional binding sites involved in the 2.8kb 5’regulatory region contains additional binding sites involved in the activation of activation of jingjing expression in trachea expression in trachea

ProximalProximal jing jing 1.3 kb 5’regulatory region contains sites required for 1.3 kb 5’regulatory region contains sites required for jingjing expression in the segmental ectodermal stripes, and for midline and tracheal expression in the segmental ectodermal stripes, and for midline and tracheal expression of expression of jingjing

Trachealess and Tango activate and are required for Trachealess and Tango activate and are required for jingjing expression expression in vivoin vivo

Pointed activates and is required for Pointed activates and is required for jingjing expression expression in vivoin vivo

Integrity of the Pointed binding sites is required for Integrity of the Pointed binding sites is required for jingjing expression expression in vivoin vivo Drifter activates and is required for Drifter activates and is required for jingjing expression expression in vivoin vivo

Tyrosine kinase signaling regulates Tyrosine kinase signaling regulates jingjing expression expression

jingjing function is required for proper cell migration during tubulogenesis function is required for proper cell migration during tubulogenesis

jingjing is required autonomously for maximal is required autonomously for maximal btl btl expression during primary expression during primary branching branching

Significance of the projectSignificance of the project

FGFR and EGFR signaling

branching morphogenesis in the vertebrate lung Null mutations in the EGFR in humans - 50% reductions in branching and neonatal failure of lung maturation

In the lung bud, FGFR type 2 isoform IIIb and its ligands (FGF10, FGF7 and FGF1) – patterning and morphogenesis of the respiratory tract

Induction of the liver over lung fate – different concentrations of the FGFs

Defects in the Sonic Hedgehog-Gli and FGF pathways – adriamycin-mediated teratogenic model tracheoesophageal anomalies

Signal transduction and its transcriptional regulation control over highly migratory cells during lung development and invasive processes such as tumor formation

In vertebrates, mutations in the gli genes (jing) absence of larynx, trachea and lung

AcknowledgementsAcknowledgements

Dr. C.Pratt, Dr. A.Colavita, Dr. D.Lohnes for Dr. C.Pratt, Dr. A.Colavita, Dr. D.Lohnes for helpful advice and guidance.helpful advice and guidance.

My supervisor Dr. M.Sonnenfeld for her help My supervisor Dr. M.Sonnenfeld for her help with my project, and in particular with confocal with my project, and in particular with confocal microscopy and light microscopy, embryo microscopy and light microscopy, embryo analysis, fly genetics, and for proof-reading of analysis, fly genetics, and for proof-reading of my thesis manuscript. my thesis manuscript.

My co-workers M’omena Dawood, Danya My co-workers M’omena Dawood, Danya Alhayari, Xuetao Sun, and Lunde Huang.Alhayari, Xuetao Sun, and Lunde Huang.

Isolated in two genetic screens

Border cell migrationin ovariesD. Montell 2001

CNS axon guidanceSedaghat et al., 2002

encodes nuclear (Gli)-Kruppel protein with 3 zinc fingers

Fusion cellFusion cell

formation of cadherin-dependent intercellular adhesion unique cell-shape change doughnut-like cell

Escargot zing finger TF

Dysfusion bHLH-PAS

TF

DE-cadherin

expression

Fusion cellTrh DysStage 12

DT/esg independent activation of DE-cad

expression

RationaleRationale WWe propose a role for e propose a role for jingjing downstream of downstream of trhtrh during tracheal development during tracheal development

(Sedaghat et al., 2002)(Sedaghat et al., 2002) jingjing expression is not observed prior to that expression is not observed prior to that trhtrh in trachea in trachea JING protein is present in tracheal nuclei, coincident with TRH during JING protein is present in tracheal nuclei, coincident with TRH during

stage 10stage 10 tracheal phenotypes of homozygous tracheal phenotypes of homozygous jingjing mutations are less severe than mutations are less severe than

those of homozygous those of homozygous trhtrh mutations mutations presence of three E-box ACGTG core sites in the 5′ regulatory region of presence of three E-box ACGTG core sites in the 5′ regulatory region of

jingjing suggest that suggest that jingjing is directly is directly regulat regulated by TRHed by TRH trhtrh embryonic phenotypes are epistatic to that of embryonic phenotypes are epistatic to that of jingjing, as shown by double , as shown by double

mutant analysis. mutant analysis. jingjing mutations genetically interact with mutations in mutations genetically interact with mutations in TRHTRH target gene target gene btlbtl Based on the requirement of Based on the requirement of jingjing is for both EGFR and FGFR-dependent is for both EGFR and FGFR-dependent

MAPK activity, we propose that jing is responsive to RTK signaling MAPK activity, we propose that jing is responsive to RTK signaling (Sonnenfeld et. al., 2004) and is essential btl expression. (Sonnenfeld et. al., 2004) and is essential btl expression.

5’5’ cttctg ctgccgcctc ttttttggat aaatggatat ttatgcttcgcgtgcccgct cttctg ctgccgcctc ttttttggat aaatggatat ttatgcttcgcgtgcccgct cgtagaaaca gaaacgtcag tgcgtagaaaca gaaacgtcag tgtgcgcgcg tgtgtttgtg tggataattatgcgcgcg tgtgtttgtg tggataatta

aagcagcaac gatttcgagt acgaaatgct ttaagaccga gattaaagtt gaaggtcggtcgttgttgtt gttgcgttcg tctgtgtttg tgtgtgcatg tgtgtggaaa agcggacaggaaaacggatc ggaatcggat aagcagcaac gatttcgagt acgaaatgct ttaagaccga gattaaagtt gaaggtcggtcgttgttgtt gttgcgttcg tctgtgtttg tgtgtgcatg tgtgtggaaa agcggacaggaaaacggatc ggaatcggat

cgcctgcctg ggaacacgca tagcgttagc gttgaacgttagtataccga cgaaggctga agtcgttaag ccaaagaata cgaatacgcc agcaaaataaaagtaatcgt gcattgctcc ctctctgctg ctgcgcatgt cgcctgcctg ggaacacgca tagcgttagc gttgaacgttagtataccga cgaaggctga agtcgttaag ccaaagaata cgaatacgcc agcaaaataaaagtaatcgt gcattgctcc ctctctgctg ctgcgcatgt

ttctctgccg cctcgctgctgcagcgctgc ctcggcctgc atttttagtt tcgcccgact attacagcat gctttacattccgtttacat cacgtgttaa ataactgctg gctgtcactg ttcgctgttc gcgaccgtgtgtgtgtaatt ttctctgccg cctcgctgctgcagcgctgc ctcggcctgc atttttagtt tcgcccgact attacagcat gctttacattccgtttacat cacgtgttaa ataactgctg gctgtcactg ttcgctgttc gcgaccgtgtgtgtgtaatt

gtgtgcaatt gttgttgttt gcccaagcaa atatgcaatg atttttgcgccacccccaac ccccatgcca tctctccccc tgcaaccagt gccttcttca ttcatgccttgcgtgtatgt gtgtgtgtgt tggatacaca cccccacatc gtgtgcaatt gttgttgttt gcccaagcaa atatgcaatg atttttgcgccacccccaac ccccatgcca tctctccccc tgcaaccagt gccttcttca ttcatgccttgcgtgtatgt gtgtgtgtgt tggatacaca cccccacatc

cgacccacgc acacaaacacacgacaaagg caacttctgg cggatgagga agccgctttc ggtgtgtgcg tgtgtatctaatgggatttt ctaataaaat ttaaattgtt gccgcttttg ccgtgcggcg cgacccacgc acacaaacacacgacaaagg caacttctgg cggatgagga agccgctttc ggtgtgtgcg tgtgtatctaatgggatttt ctaataaaat ttaaattgtt gccgcttttg ccgtgcggcg

ttcgttctatgaatttaata tgacccctgt gtgtgtgaat aggggccata aagctttgct tatggctggaaaattaaata gagtgcataa tttaagcatc aactttgcgt tgaatgcata ataaacggctgcaattgaaa ttcgttctatgaatttaata tgacccctgt gtgtgtgaat aggggccata aagctttgct tatggctggaaaattaaata gagtgcataa tttaagcatc aactttgcgt tgaatgcata ataaacggctgcaattgaaa

ggcgccgtct accaaaggac tactgcataa atatcggata acttatactcgggctcagtg ccaccagatt cacaatttgc aacggacgaa gggaatgtaa gtccctggaaatggccgtcg gcaaacagaa agtgtctgat ggcgccgtct accaaaggac tactgcataa atatcggata acttatactcgggctcagtg ccaccagatt cacaatttgc aacggacgaa gggaatgtaa gtccctggaaatggccgtcg gcaaacagaa agtgtctgat

tatgtcgttc aaaatgttag ctatattaactgccagtcaa cgatacaatg tgtagtttaa agctgtcacg attttcgcag tgcgtctgtcaattaatgag cttttcaaat atataaatat tagttccccg aaaaaatttc tatgtcgttc aaaatgttag ctatattaactgccagtcaa cgatacaatg tgtagtttaa agctgtcacg attttcgcag tgcgtctgtcaattaatgag cttttcaaat atataaatat tagttccccg aaaaaatttc

aacagtaagggatgcatacg ggtgatttaa aattacttaa aagaaatcat tctgcttaaa tttatatgctttaaaccaac attacgtgat ttcaaattta aaaaataata gtgaatacca ctttacgatagcagagctct aacagtaagggatgcatacg ggtgatttaa aattacttaa aagaaatcat tctgcttaaa tttatatgctttaaaccaac attacgtgat ttcaaattta aaaaataata gtgaatacca ctttacgatagcagagctct

caattaaacc ctactgtttt aaaacaagtg agtgtgtaca cacatatactgatggtattg atgtttgttt ataaaacgta ctccatatta aacattactt gactacgtgtccctcaattt gttgttagaa attaaacgaa atttgagtcc caattaaacc ctactgtttt aaaacaagtg agtgtgtaca cacatatactgatggtattg atgtttgttt ataaaacgta ctccatatta aacattactt gactacgtgtccctcaattt gttgttagaa attaaacgaa atttgagtcc

aacagatttc tgtgtcataaacagatttc tgtgtcatatttgagtatga atccgcatct gtttgagtatga atccgcatct gtaaaaaaaa agcagcgcta aatgcacgaa aacaaaaatggtcggggtcg tccaccccat taaaaaatta cacaaatgca ggagaaaaat taaaaaaaa agcagcgcta aatgcacgaa aacaaaaatggtcggggtcg tccaccccat taaaaaatta cacaaatgca ggagaaaaat

ggacgtaataggcttatcac aaaagcagtt tggtcgtcgt aatctgtggt atctcccggg gtaggcaaacataaaataaa atgtgttttc atttcatgag ttttcttttt gttgtgcttt gtacatagatctaaaatttg agtaggtaga ggacgtaataggcttatcac aaaagcagtt tggtcgtcgt aatctgtggt atctcccggg gtaggcaaacataaaataaa atgtgttttc atttcatgag ttttcttttt gttgtgcttt gtacatagatctaaaatttg agtaggtaga

acttggatgt gattaaaaat gtttcacgga aggccgacggaaaatacaaa accctgaaaa tcgctgtcca caaagatttc gggtctagaa tccaagctcgactaaacgat ttattttaac gatcgagtaa agcgtctcta acttggatgt gattaaaaat gtttcacgga aggccgacggaaaatacaaa accctgaaaa tcgctgtcca caaagatttc gggtctagaa tccaagctcgactaaacgat ttattttaac gatcgagtaa agcgtctcta

tagctaaaga ttaaaaaaaatattttttac atatttgttt gcaattgcaa cttctacata tgtacatatg tgaaagacaatatgtagaaa gcacgcttgc ttcgttttag ttattcaatt gatggataaa tggcattgtatttattctgc tagctaaaga ttaaaaaaaatattttttac atatttgttt gcaattgcaa cttctacata tgtacatatg tgaaagacaatatgtagaaa gcacgcttgc ttcgttttag ttattcaatt gatggataaa tggcattgtatttattctgc

acttatgtat tgtatattta aagtttttag cgttttaaat tcattttttaataagcttgt aaacttttgc gtgttccaaa agtgaaaaag tatcatctat ttgtatatcttttcgagcac caaaatagca gaccgaattc aaaaataccc acttatgtat tgtatattta aagtttttag cgttttaaat tcattttttaataagcttgt aaacttttgc gtgttccaaa agtgaaaaag tatcatctat ttgtatatcttttcgagcac caaaatagca gaccgaattc aaaaataccc

tttttgtgta accagaggattttttgtata cttgccgacg tttatcccgt ttcacatgta catgtgttta accgaaaccgcacaaacaca cacgcgactt ggggctacct gaaactgaaa ttttcccgca ttagattgcggcccttgcct tttttgtgta accagaggattttttgtata cttgccgacg tttatcccgt ttcacatgta catgtgttta accgaaaccgcacaaacaca cacgcgactt ggggctacct gaaactgaaa ttttcccgca ttagattgcggcccttgcct

tccatgcttc gtcttcttct tcttctttct catcgttgtt tctgtaaataaagaaagatg tggagaggga gttttttctt cctttacgtt cgctctttct tccagttttttttgtttact ttttattact tttcatgttc tcttccacga aaattagtgc tccatgcttc gtcttcttct tcttctttct catcgttgtt tctgtaaataaagaaagatg tggagaggga gttttttctt cctttacgtt cgctctttct tccagttttttttgtttact ttttattact tttcatgttc tcttccacga aaattagtgc

gaaacagaaacgaattgctt agcgtttatc gacccgtttt gtgttctctt attctgttgc gctcttctccttctcttgtt cgctctcttg aaacaataat ttccctattt ctcgtgtaaa atgaatacatgttttttaca gttattcaaa gaaacagaaacgaattgctt agcgtttatc gacccgtttt gtgttctctt attctgttgc gctcttctccttctcttgtt cgctctcttg aaacaataat ttccctattt ctcgtgtaaa atgaatacatgttttttaca gttattcaaa

attaaaatat tttgtatcac aaacacggag taaatcgattattttaagat taattttaaa aatgttttgc gcgcatgttg ttgaatttaa tttcgcagctgaatgccttg tgagttctct tattctctttattaaaatat tttgtatcac aaacacggag taaatcgattattttaagat taattttaaa aatgttttgc gcgcatgttg ttgaatttaa tttcgcagctgaatgccttg tgagttctct tattctcttt agctcttctc cttctcttgt agctcttctc cttctcttgt

tctcggtctcttgcaaccatat tttccttatt tctcgtgtaa aatgaatata tgttttttta cagtttctctaaattaaaat aatttctatc acatcatcga gtaaatcgat tttttaaaga ttaattttaaaattgttgct tgcgcgcctc ttattgaatt ggtctcttgcaaccatat tttccttatt tctcgtgtaa aatgaatata tgttttttta cagtttctctaaattaaaat aatttctatc acatcatcga gtaaatcgat tttttaaaga ttaattttaaaattgttgct tgcgcgcctc ttattgaatt

tagtttcata gctgcttgcc ttttactgcgggttgttttg ggaaatttat cagctgttag ccgcgctgtt agaggtaaca gcgcttgcgacggacattct ctaacacagc cgggaaataa acatccaaga ataatttgag tagtttcata gctgcttgcc ttttactgcgggttgttttg ggaaatttat cagctgttag ccgcgctgtt agaggtaaca gcgcttgcgacggacattct ctaacacagc cgggaaataa acatccaaga ataatttgag

tggccttccaacactgccagtggccttccaacactgccag gcaaataaac gcaaataaac 3’3’

2.8kb1.5kb1.3kb

1.5j

ingΔ

Pn

tΔA

op-l

acZ

jjinging1.51.5-lacZ-lacZ expression is responsive to FGFR and expression is responsive to FGFR and EGFR signaling in trachea and is regulatedEGFR signaling in trachea and is regulated by by

Trachealess and DrifterTrachealess and Drifter (stage 12-15) (stage 12-15)..

0

20

40

60

80

100

120

1.5ji

ng la

cZ/D

fr/Dfr

1.5ji

ng la

cZ/bn

l/bnl

1.5ji

ng la

cZ/rh

o/rh

o

1.5ji

ng la

cZ/tr

h/trh

1.5ji

ng la

cZ/pn

t/pnt

deletio

n pnt

1.5ji

ng la

cZ

DB

DT

LT

% o

f se

gmen

ts e

xpre

ssin

g ji

ng1

.5-l

acZ

jingjing expression is regulated by expression is regulated by Dfr in vivoDfr in vivoStable line 3: [jing1.5-lacZ/jing1.5-lacZ];

[DfrE082/DfrE082]

control jing1.5-lacZ

dor

sal

dor

sal

sagi

ttal

A A’ A’’A’’

B ’’

C C’ C’’

ATGGATATTTATGAATGGATATTTATGCTTCGCGTGCCCGCTCGTAGAAACAGAAACGTCAGTGTGCGCGCGTGTGTTTGTGTGGATAATTAAAGCAGCAACGATTTCGAGTACGAAATGCTTTAAGACCGAGATTAAAGTTGAAGGTCGGTCGTTGTTGTTGTTGCGTTCGTCTGTGTTTGTGTGTGCATGTGTGTGGAA

AAGCGGACAGGAAAACGGATCGGAATCGGATCGCCTGCCTGGGAACACGCATAGCGTTAGCGTTGAACGTTAGTATACCGACGAAGGCTGAAGTCGTTAAGCCAAAGAATACGAATACGCCAGCAAAATAAAAGTAATCGTGCATTGCTCCCTCTCTGCTGCTGCGCATGTTTCTCTGCCGCCTCGCTGCTGCAGCGCT

GCCTCGGCCTGCATTTTTAGTTTCGCCCGACTATTACAGCATGCTTTACATTCCGTTTACATCACGTGTTAAATAACTGCTGCCTGTCACTGTTCGCTGTTCGCGACCGTGTGTGTGTAATTGTGTGCAATTGTTGTTGTTTGCCCAAGCAAATATGCAATGATTTTTGCGCCACCCCCAACCCCCATGCCATCTCTCCCCCTG

CAACCAGTGCCTTCTTCATTCATGCCTTGCGTGTATGTGTGTGTGTGTTGGATACACACCCCCACATCCGACCCACGCACACAAACACACGACAAAGGCAACTTCTGGCGGATGAGGAAGCCGCTTTCGGTGTGTGCGTGTGTATCTAATGGGATTTTCTAATAAAATTTAAATTGTTGCCGCTTTTGCCGTGCGGCGTTC

GTTCTATGAATTTAATATGACCCCTGTGTGTGTGAATAGGGGCCATAAAGCTTTGCTTATGGCTGGAAAATTAAATAGAGTGCATAATTTAAGCATCAACTTTGCGTTGAATGCATAATAAACGGCTGCAATTGAAAGGCGCCGTCTACCAAAGGACTACTGCATAAATATCGGATAACTTATACTCGGGCTCAGTGCCA

Pnt AOP/YanCCAGATTCACAATTTGCAACGGACGAAGGGAATGTAAGTCCCTGGAAATGGCCGTCGGCAAACAGAAAGTGTCTGATTATGTCGTTCAAAATGTTAGCT

ATATTAACTGCCAGTCAACGATACAATGTGTAGTTTAAAGCTGTCACGATTTTCGCAGTGCGTCTGTCAATTAATGAGCTTTTCAAATATATAAATATTAGTTCCCCGAAAAAATTTCAACAGTAAGGGATGCATACGGGTGATTTAAAATTACTTAAAAGAAATCATTCTGCTTAAATTTATATGCTTTAAACCAACATT

ACGTGATTTCAAATTTAAAAAATAATAGTGAATACCACTTTACGATAGCAGAGCTCTCAATTAAACCCTACTGTTTTAAAACAAGTGAGTGTGTACACAC

ATATACTGATGGTATTGATGTTTGTTTATAAAACGTACTCCATATTAAACATTACTTGACTACGTGTCCCTCAATTTGTTGTTAGAAATTAAACGAAATTTGAGTCCAACAGATTTCTGTGTCATATTTGAGTATGAATCCGCATCTGTAAAAA

CME1

CME2

CME3

CMEs

1(2)01094

A

B

mut5

Jing regulatory region Sedaghat Y. et al., 2002

2.8kb2.8kb

lacZ reporter constructs

Adapted from Sedaghat et al., 2002

0

10

20

30

40

50

60

jing1.5-lacZ jing1.5-lacZ/rho

DB weak

DT weak

LT weak

% of the segments showing weak lacZ expression

jing1.5-lacZ jing1.5-lacZ/rho

Ectopic expression of Ectopic expression of jingjing in a pair-rule pattern in a pair-rule pattern induced by Trh and Dfr / induced by Trh and Dfr / prdprd--Gal4Gal4; UAS-; UAS-trhtrh, , vvlvvl/ /

jing1.5-lacZjing1.5-lacZ / mouse anti- / mouse anti--gal antibody-gal antibody

prd-Gal4; UAS-vvl,trh

control, btl probe

ven

tral

control jing1.5-lacZ

BC

A

prd-Gal4; UAS-vvl,trh/jing1.5-lacZ

stage 12 stage 12

c. Ectopic activation of c. Ectopic activation of jing1.5-jing1.5-lacZlacZ by using UAS/ by using UAS/Gal4Gal4 system system

b. Generation of stable linesb. Generation of stable lines

a. a. in situin situ hybridization with hybridization with jingjing RNA probeRNA probe

d. Generation of d. Generation of jing1.5ΔPnt-lacZ jing1.5ΔPnt-lacZ construct and expression pattern of construct and expression pattern of

jing1.5ΔPnt-lacZjing1.5ΔPnt-lacZ reporter reporter

1. Generation of 1.5 kb, 1.3 kb and 1. Generation of 1.5 kb, 1.3 kb and 2.8 kb 2.8 kb lacZ lacZ reporter constructsreporter constructs

jingjing is required to maintain MAPK activity in the is required to maintain MAPK activity in the trachea and CNS midlinetrachea and CNS midline

Adapted from Sonnenfeld M. et al., 2004

jing3 – strong hypomorph

Rationale

Genetic interactions in the tracheaGenetic interactions in the trachea

A L

Adapted from Sedaghat Y. et al., 2002

Loss of tracheal tubules

jingjing expression is not observed prior to expression is not observed prior to trhtrh

JING protein is present in tracheal nuclei coincident with JING protein is present in tracheal nuclei coincident with TRH during stage 10TRH during stage 10

tracheal phenotypes of homozygous tracheal phenotypes of homozygous jingjing mutations are mutations are less severe than those of homozygous less severe than those of homozygous trhtrh mutations mutations

three E-box ACGTG core sitesthree E-box ACGTG core sites in the 5′ regulatory regionin the 5′ regulatory regionof of jingjing directly directly regulat regulated by TRHed by TRH

trhtrh embryonic phenotypes are epistatic to that of embryonic phenotypes are epistatic to that of jingjing ((double mutant analysisdouble mutant analysis))

jingjing mutations genetically interact with mutations in mutations genetically interact with mutations in TRHTRH target gene target gene btlbtl

Stable lines Stable lines

jing1.5-lacZ/jing1.5-lacZ 2nd chromosome

pnt/TM3ubx-lacZ3rd chromosome

progeny

[jing1.5-lacZ/+ ];[pnt/+] long hair adults

[jing1.5-lacZ/+];[TM3ubx-lacZ/+]

stubbled adults

jing1.5-lacZ/jing1.5-lacZ; pnt/TM3ubx-lacZ

stubbled, tested by complementation

X

X

1. jing1.5-lacZ;trh2/TM3ubx-lacZ2. jing1.5-lacZ;pntE039/TM3ubx-lacZ3. jing1.5-lacZ;dfrE082/TM3ubx-lacZ4. jing1.5-lacZ;rho7M/TM3ubx-lacZ 5. jing1.5-lacZ;bnl1/TM3ubx-lacZ

6. jing1.5-lacZ;rhum3/rhum3