Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 1
1
Prof. Rolf Bodmer
Burnham Institute for Medical Research
La Jolla, CA
Development and Physiology
of the Heart
“The Legacy of Drosophila Genetics”
2
The Drosophila heart is a simple tube
Bodmer, 1995, TCM; Bier and Bodmer, 2004
rg lg
e
pc cc
am
3
The homeobox transcription factor, Tinman, is required for heart development
Bodmer et al., 1990; Bodmer, 1993; Venkatesh et al., 2000
Tinman mutants
have no heart
wt
tin-
Tinman
The homeobox transcription factor, Tinman, is required for heart development
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 2
4
Twist
tin RNA
Gastrulation, mesoderm movement and tinman expression
Bodmer, 1995; Lockwood and Bodmer, 2002
Gastrulation, mesoderm movement and tinman expression
5
Embryonic fly heart
Embryonic mouse heart
6
The embryology of heart formation
is remarkably similar
between flies and vertebrates:
Flies
• Both derive from mesoderm
distal to the point of gastrulation
(dorsal in vertebrates, ventral
in flies)
• Both form a linear primitive heart
tube from bilateral primordia
distal to the gut and CNS
• Both vertebrate and fly hearts
require the function of tinman-
type homeobox genes
Bodmer, 1995
Vertebrates
D Dcmvm
sm
cns
vm
ht
sm
cmV V
A
A
ht
P
P
vmpmc
eccns
ht
ecpmc
ht
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 3
7
tinman
The hearts of Drosophila and vertebrates originate from equivalent embryological positions
Nkx2.5
T. Schultheiss
Lockwood and Bodmer, 2002; Bier and Bodmer, 2004
8
tinman expression
during Amphioxu s heart
development occurs
only after the bilateral
visceral mesoderm
primordia of the lateral
plate mesoderm
have fused
at the ventral midline
Have the bilaterally
symmetrical cardiac
primordia in chordates
and arthropods evolved
independently?
Holland et al., 2003
vp
ppvm
vppp
pvc
vp
pp
pvcsev
bl
9
Mesoderm
twist tinman
Dorsal Mesoderm Heart Precursors
dpp (TGF-β )β )β )β ) wingless (Wnt)
Genetic control of heart development
Lockwood and Bodmer, 2002
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 4
10
How is the heart positioned correctly?
dppwg
tin
Lockwood and Bodmer 2002
11Lockwood and Bodmer, 2002
Do wg and dpp provide instructive information to position the heart?
dpp
st 9
wg tinman
st 10
dpp
st 10 st 11
tin
st 11 st 12
wg dpp
tin
12
UASwg
UASdpp
UASdpp; UASwg
twi24B-Gal4 X UAS-lacZ
UAS-GAL4 Mis-Expression System
Lockwood and Bodmer, 2002
Brand and Perrimon, 1993, Development
Enhancer Trap GAL4 UAS-Gene X
X
Genomic Enhancer
Tissue-specific expression of GAL4 Transcriptional activation of Gene X
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 5
13
Stage 11 tinman pattern occurs only in regions
exposed to wg and dpp at stage 9
Lockwood and Bodmer, 2002
stage 9 wg + dpp
wildtype
stage 11 tin
wgdpp
wg + dpp
twi24B>wg
twi24B>dpp
twi24B>dpp/wg
14
Stage 12 tinman pattern occurs only in regions exposed
to both wg and dpp at both stage 9 and stage 11
twi24B>dpp/wg
twi24B>dpp
twi24B>wg
stage 11wg + dpp
stage 9wg + dpp
wildtype
stage 12 tin
15
dpp wg tin tin
tin
stage 9 stage 10
stage 12stage 11
dppI
II
I
II
CMCardiac
Cell
Fates
stage 11
The changing pattern of dpp and wgexpression predicts tinman expression
Lockwood and Bodmer, 2002; Bodmer and Frasch, 1999
dpp, wg
dpp, wg
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 6
16
dpp; wg dpp; wg
twist
Mesoderm
tinman
Dorsal Mesoderm
wg and dpp signaling in the context of tinman specifies cardiac positioning and differentiation
tinman
Heart Precursors
Lockwood and Bodmer, 2002
17
Dorsal Mesoderm
pannier
twist
Mesoderm
dpp; wgdpp; wg
Cardiac Mesoderm
tinman tinman
tinman
Ectoderm
Combinatorial specification of the Drosophilaheart progenitors
Prc/DMef2
Heart
II. Morphogenesis
I. Cell type diversification
neuromancer
18
wt
neuromancer/Tbx20 is required for cell polarity
independently from the alignment of the myocardial
cells (red) at the midline
Qian et al., 2005, Dev Biol
nmr614;24B>nmr2-RN Ai
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 7
19
slit is expressed
in the Drosophila heart
nmr-lacZ
Slit
nmr-lacZ Slit
Slit is conserved and provides cues
for axon tract and muscle positioning
Qian et al., 2005, Current Biology
Wild type Slit
20
Myocardial alignment and cardiac cell polarity
is compromised in slit mutants after the bilateral rows
of progenitors meet
Crb
Crumbs and ectodermal dorsal closure occurs normally in slit mutants
Qian et al., 2005, Current Biology
Before closure
After closure
21Qian et al., 2005, Current Biology
Before reaching
dorsal midline
After reaching
dorsal midline
Polarity feature
Wild type
slit LOF
Polarity feature
MC PC Robo2 Slit/Robo Polarity markers (Dlg, α-Spectrin, etc.)
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 8
22Tin Eve
Han and Bodmer, 2003
• How is the heart
field subdivided ?
• How are individual
progenitor cells
positioned?
dpp; wgdpp; wg
Cardiac Mesoderm
tinman
Ectoderm
pannier
neuromancertwist
Mesoderm Dorsal Mesoderm
tinman tinman
23
eme900 - LacZ Eve eme900-Gal4>GFP
Integration of positional, tissue-context and lineage information at the transcriptional
level: regulation of the mesodermal even-skipped enhancer (eme)
eme-6.4kb
900bp
Eve
protein coding
+9.2kb
Fujioka et al., 2005
24
LacZ (red)
Eve & LacZ (yellow)
Eve (green)
emeA (220-600)
++
Transcription factor consensus binding sites of known cardiac regulators are concentrated
in eve’s mesodermal enhancer (eme)
Consensus binding sites: Tinman dTCF (wg)
Ladybird Zfh-1 Su(H) (Notch)
Medea(dpp)
eme900
Han et al., 2002
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 9
25
Dpp Wg
Tkv/Pnt
Mad/Medearas
PntP2
Dfz2
Arm/dTCF
Dpp
Pnr. Ush
Tin
Lb
Twi
eme
ectoderm
mesoderm
How are the mesodermal Eve progenitors specified as a small subset of the cardiac mesoderm?
Tin Eve Lb Eve
eve expression within cardiac mesoderm
Ladybird (Lb, a homeobox transcription factor)
26
Ladybird and Even-skipped are mutual repressors
wt
eme>Lbe
Mef2 (green) Eve (red)
EPC progenitor-
specific expression
of ladybird
suppresses
Eve expression
Han et al., 2002
Mef2 (green) Eve (red)
27
-6.4kbEve
+8.2kb
Ladybird and Even-skipped are mutual repressors
In the absence of mesodermal even-skipped expressio n
ladybird expands into the Eve territoryHan et al., 2002
wt
Eve Eve
emeA-LacZ P[eve-eme-],eveR13; emeA
Lbe LacZ
Ladybird and Even-skipped are mutual repressors
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 10
28
Dpp
TinMad/Medea
Wg
Dfz2Tkv/Pnt
ectoderm
mesoderm
eme
���� Eve progenitors within cardiac mesoderm
Arm/dTCF Twi
Lb
ras
MAPK/ETS
Tin Eve
Pnr, Ush
Dpp
??
29
Hedgehog promotes specification of Eve progenitors and inhibits Lbe expression
Lbe Eve
wt
Eve Hh
wt
meso>wg, hh13c
Lbe Eve Lbe Eve
Meso>hh
Liu et al., 2006, Dev Biol
Hedgehog promotes specification of Eve progenitors and inhibits Lbe expression
30
Model: Hedgehog (Hh) sets up a gradient of RTK signaling
within the cardiogenic mesoderm to determine cell fates
along the anterior-posterio r axis:
���� High levels of ras signaling specifies Eve cells
���� Low levels of ras signaling specifies Lbe cells
Eve Hh
L
Lbe Eve
Hh EGF (spitz/vein)
rho
EGR-R
Ras
Lbe [--] Eve Ets (pointed)
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 11
31
Combinatorial specification of the Drosophilaheart progenitors
Eve Hh
I. Cell type diversification
II. Morphogenesis
Prc/DMef2
Heart
twist
Mesoderm
dpp; wgdpp; wg
Cardiac Mesoderm
Dorsal Mesoderm
tinman tinman
tinman
Ectoderm
pannier
neuromancer
32
Adult
Heart
Monier et al., 2005, Development
Posterior aorta
Larva
A5 A6-A7
Heart
A1-A4
Svp Tin Tin/Ih/Ork1
33
Image-based Analysis of Heart Function
Semi-intact Drosophila Preparation
Ocorr et al., 2007, PNAS
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 12
34
Image-based Analysis of Heart Contractions
• Define a 1 pixel wide SLICE
• Identical slices “CUT” out of each movie frame
• Each Slice is ALIGNED Horizontally
• M-mode trace
Ocorr et al., 2007, PNAS
35
KCNQ Genes
• 1 KCNQ gene in Drosophila
5 KCNQ genes in humans
• Mutations in KCNQ genes result in Cardiac
Arrhythmias & Long QT syndrome KCNQ Genes
36
Ca2+IK
+
Ca2+
/ Na+
IKs
Threshold
IKr
KCNQ
I leak K+ I pacemaker
I V-D
cation
Potassium channels responsible for IKS in human heart muscle
(repolarizes the cardiac action potential)
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 13
37
1 week KCNQ 186 mutant
5 week KCNQ 186 mutant
Rhythmicity Abnormalities in KCNQ Mutants
1 weekWild type
Meso>KCNQ;KCNQ 186���� “rescue”
Ocorr et al., PNAS (2007)
5 weekWild type
38
wt
KCNQ
“Index of Arrhythmia”: standard deviation of the normalized heart period
Ocorr et al., 2007, PNAS
Heart Period“Index of Arrhythmia”: standard deviation of the normalized heart period
39
Distribution of systolic intervals broadens with age
and is accelerated in KCNQ mutants
Arrhythmias that develop with age are rescued with KCNQ overexpression
Ocorr et al., 2007, PNAS
KCNQ97
KCNQ186Distribution of systolic intervals broadens with age
and is accelerated in KCNQ mutants
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 14
40
A cardiac stress test using a pacemaker to assess heart performance
Wessells and Bodmer, 2004
Does the heart become dysfunctional ?= arrest or fibrillation “electrical failure”
Normal heart rate: 3Hz
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 s
Pacing the heart (40V): 6 Hz for 30 sec
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 s
41
Mutations in KCNQ potassium channel increase pacing-induced heart failure
Ocorr et al., 2007, PNAS
yw w KCNQ mutant 1
KCNQ mutant 2
KCNQ mutant 1/
24B, UAS KCNQ
KCNQ mutant 1/
24B, UAS KCNQ
Heart failure rate1 week old flies
42
Electrical pacing
6 Hz for 30s
Pacing-induced heart failure increases with age
Wessells et al., 2004
Decline of cardiac function with age
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 15
43Garofalo, 2002
Insulin receptor
(InR) pathway
is a highly
conserved regulator
of growth and life
span in flies,
worms and mice
How is the fly’s heart
function affected
by the InR pathway?
(Foxo)
(PKB)
(Foxo)
44
Insulin receptor substrate
homolog mutants (chico)
prolong life-span in flies
(Clancy et al., 2001)
*
chico mutants
• live longer
• old hearts fail
as little
as young hearts
Wessells et al., 2004
Tu et al. 2002 Aging Cell
Age (days)
ch/+
ch/ch
+/+
Survival (lx)
45
The age-dependent increase in electrically induced
heart failure rate is similar between males or females
Wessells et al., 2004
Failure rate
Age (weeks)
Failure rate
Age (weeks)
Cardiac aging is not observed in chico or InR mutants
Development and Physiology of the Heart
“The Legacy of Drosophila Genetics”
Prof. Rolf Bodmer
The screen versions of these slides have full details of copyright and acknowledgements 16
46
UAS-dPTEN x GMH5UAS-dfoxo(3x) x GMH5
UAS-InR x GMH5
Wessells et al., 2004
Does heart-specific overexpression of components
of the InR signaling pathway autonomously affect
cardiac aging ?
The age-dependent increase in pacing-induced heart failure rate
is abolished with heart-specific overexpression of InR signaling:
• Pathway activation prematurely increases the risk of heart failure
• Pathway inhibition lowers the risk of heart failure at older ages
FOXO
47
Insulin – TOR signaling pathways autonomously regulate heart function in aging Drosophila
48
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