Development and Physiology of the Heart “The Legacy of ...

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


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



Prof. Rolf Bodmer


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



Prof. Rolf Bodmer


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



Prof. Rolf Bodmer


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


Brand and Perrimon, 1993, Development

Enhancer Trap GAL4 UAS-Gene X

X

Genomic Enhancer

Tissue-specific expression of GAL4 Transcriptional activation of Gene X



Prof. Rolf Bodmer


13

Stage 11 tinman pattern occurs only in regions

exposed to wg and dpp at stage 9


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



Prof. Rolf Bodmer


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


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



Prof. Rolf Bodmer


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.)



Prof. Rolf Bodmer


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



Prof. Rolf Bodmer


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


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




Prof. Rolf Bodmer


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)



Prof. Rolf Bodmer


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



Prof. Rolf Bodmer


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


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)



Prof. Rolf Bodmer


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


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


KCNQ97

KCNQ186Distribution of systolic intervals broadens with age

and is accelerated in KCNQ mutants



Prof. Rolf Bodmer


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


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



Prof. Rolf Bodmer


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


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


Failure rate

Age (weeks)

Failure rate

Age (weeks)

Cardiac aging is not observed in chico or InR mutants



Prof. Rolf Bodmer


46

UAS-dPTEN x GMH5UAS-dfoxo(3x) x GMH5

UAS-InR x GMH5


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

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Development and Physiology of the Heart “The Legacy of ...

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