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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