Neural

Systems

Responding

to

Degrees

of

Uncertainty

in

Human

Decision-Making

Ming

Hsu

et

al.

Science

-LRB-

-RRB-

DOI

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REPORTS

Fig.

Field

emission

scanning

electron

microscopy

-LRB-

FESEM

-RRB-

of

cerebellar

granular

neurons

exposed

to

taipoxin

-LRB-

nM

for

min

-RRB-

or

mLysoPC

OA

-LRB-

mM

for

min

-RRB-

at

lower

-LRB-

left

panels

-RRB-

and

higher

-LRB-

right

panels

-RRB-

magnifications

-LRB-

A

-RRB-

Identical

results

were

obtained

with

notexin

b-bungarotoxin

and

textilotoxin

Scale

bar

mm

-LRB-

left

panels

-RRB-

and

mm

-LRB-

right

panels

-RRB-

-LRB-

B

-RRB-

Cerebellar

neurons

were

exposed

to

nM

b-bungarotoxin

for

min

or

to

mM

mLysoPC

OA

for

min

and

stained

with

an

antibody

specific

for

the

lumenal

domain

of

synaptotagmin

I

before

fixation

Samples

were

processed

for

indirect

immunofluorescence

without

permeabilization

superimposable

results

were

obtained

with

notexin

taipoxin

and

textilotoxin

in

cerebellar

neurons

and

hippocampal

neurons

Scale

bar

mm

I.

L.

Chen

C.

Y.

Lee

Virchows

Arch

B

Cell

Pathol

-LRB-

-RRB-

J.

B.

Harris

B.

D.

Grubb

C.

A.

Maltin

R.

Dixon

Exp

Neurol

-LRB-

-RRB-

C.

Y.

Lee

M.

C.

Tsa

Y.

M.

Chen

A.

Ritonja

F.

Gubensek

Arch

Int

Pharmacodyn

Ther

-LRB-

-RRB-

P.

Rosenberg

Venom

Phospholipase

Enzymes

R.

M.

Kini

Ed

-LRB-

Wiley

Chichester

UK

-RRB-

pp.

R.

M.

Kini

Toxicon

-LRB-

-RRB-

C.

C.

Yang

in

Venom

Phospholipase

Enzymes

R.

M.

Kini

Ed

-LRB-

Wiley

Chichester

UK

-RRB-

pp.

R.

E.

Stafford

T.

Fanni

E.

A.

Dennis

Biochemistry

-LRB-

-RRB-

J.

Wang

et

al.

Br

J.

Pharmacol

-LRB-

-RRB-

M.

Rigoni

et

al.

J.

Cell

Sci

-LRB-

-RRB-

D.

Bonanomi

et

al.

Mol

Pharmacol

-LRB-

-RRB-

F.

Kamp

D.

Zakim

F.

Zhang

N.

Noy

J.

A.

Hamilton

Biochemistry

-LRB-

-RRB-

L.

V.

Chernomordik

E.

Leikina

V.

Frolov

P.

Bronk

J.

Zimmerberg

J.

Cell

Biol

-LRB-

-RRB-

L.

V.

Chernomordik

M.

M.

Kozlov

Annu

Rev.

Biochem

-LRB-

-RRB-

Y.

Xu

F.

Zhang

Z.

Su

J.

A.

McNew

Y.

K.

Shin

Nat

Struct

Mol

Biol

-LRB-

-RRB-

C.

G.

Giraudo

et

al.

J.

Cell

Biol

-LRB-

-RRB-

C.

Reese

F.

Heise

A.

Mayer

Nature

-LRB-

-RRB-

K.

Farsad

P.

De

Camilli

Curr

Opin

Cell

Biol

-LRB-

-RRB-

R.

Jahn

T.

Lang

T.

C.

Sudhof

Cell

-LRB-

-RRB-

L.

K.

Tamm

J.

Crane

V.

Kiessling

Curr

Opin

Struct

Biol

-LRB-

-RRB-

E.

R.

S.

Roldan

Front

Biosci

-LRB-

-RRB-

S.

Wei

et

al.

Neuroscience

-LRB-

-RRB-

Supported

by

Telethon

grant

COFIN

Project

FISR-DM

University

of

Padova

and

Cancer

Research

UK

Supporting

Online

Material

Materials

and

Methods

Figs

and

References

September

accepted

November

science

ment

of

in

other

exocytotic

events

such

as

the

sperm

acrosomal

exocytosis

-LRB-

-RRB-

Furthermore

a

SPAN

microinjected

into

pheochromocytoma

cells

inhibited

neuroexocytosis

-LRB-

-RRB-

presumably

because

it

acted

on

the

cytosolic

plasma

membrane

side

inducing

an

opposite

membrane

configuration

The

presence

of

clathrin-coated

W-shaped

structures

in

SPANpoisoned

NMJs

-LRB-

-RRB-

suggested

that

they

also

inhibit

synaptic

vesicle

fission

from

the

plasma

membrane

-LRB-

-RRB-

Indeed

the

same

SPAN

induced

lipid

changes

promoting

membrane

fusion

do

inhibit

membrane

fission

for

the

same

physical

and

topological

reasons

-LRB-

-RRB-

References

and

Notes

R.

M.

Kini

Ed.

Venom

Phospholipase

Enzymes

-LRB-

Wiley

Chichester

UK

-RRB-

G.

Schiavo

M.

Matteoli

C.

Montecucco

Physiol

Rev.

-LRB-

-RRB-

C.

Montecucco

O.

Rossetto

Trends

Biochem

Sci

-LRB-

-RRB-

S.

G.

Cull-Candy

J.

Fohlman

D.

Gustavsson

R.

LullmannRauch

S.

Thesleff

Neuroscience

-LRB-

-RRB-

Neural

Systems

Responding

to

Degrees

of

Uncertainty

in

Human

Decision-Making

Ming

Hsu

Meghana

Bhatt

Ralph

Adolphs

Daniel

Tranel

Colin

F.

Much

is

known

about

how

people

make

decisions

under

varying

levels

of

probability

-LRB-

risk

-RRB-

Less

is

known

about

the

neural

basis

of

decision-making

when

probabilities

are

uncertain

because

of

missing

information

-LRB-

ambiguity

-RRB-

In

decision

theory

ambiguity

about

probabilities

should

not

affect

choices

Using

functional

brain

imaging

we

show

that

the

level

of

ambiguity

in

choices

correlates

positively

with

activation

in

the

amygdala

and

orbitofrontal

cortex

and

negatively

with

a

striatal

system

Moreover

striatal

activity

correlates

positively

with

expected

reward

Neurological

subjects

with

orbitofrontal

lesions

were

insensitive

to

the

level

of

ambiguity

and

risk

in

behavioral

choices

These

data

suggest

a

general

neural

circuit

responding

to

degrees

of

uncertainty

contrary

to

decision

theory

In

theories

of

choice

under

uncertainty

used

in

social

sciences

and

behavioral

ecology

the

only

variables

that

should

influence

an

uncertain

choice

are

the

judged

probabilities

of

possible

outcomes

and

the

evaluation

of

those

outcomes

But

confidence

in

judged

probability

can

vary

widely

In

some

choices

such

as

gambling

on

a

roulette

wheel

probability

can

be

confidently

judged

from

relative

frequencies

event

histories

or

an

accepted

theory

At

the

other

extreme

such

as

the

chance

of

a

terrorist

attack

probabilities

are

based

on

meager

or

conflicting

evidence

where

important

information

is

clearly

missing

The

two

VOL

SCIENCE

types

of

uncertain

events

are

often

called

risky

and

ambiguous

respectively

In

subjective

expected

utility

theory

the

probabilities

of

outcomes

should

influence

choices

whereas

confidence

about

those

probabilities

should

not

But

experiments

show

that

many

people

are

more

willing

to

bet

on

risky

outcomes

than

on

ambiguous

ones

holding

judged

probability

of

outcomes

constant

-LRB-

-RRB-

This

empirical

aversion

to

ambiguity

motivates

a

search

for

neural

distinctions

between

risk

and

ambiguity

Here

we

extend

the

study

of

the

neural

basis

of

decision

under

risk

to

encompass

ambiguity

The

difference

between

risky

and

ambiguous

uncertainty

is

illustrated

by

the

Ellsberg

paradox

-LRB-

-RRB-

Imagine

one

deck

of

cards

composed

of

red

and

blue

cards

-LRB-

the

risky

deck

-RRB-

Another

deck

has

red

or

blue

cards

but

the

composition

of

red

and

blue

cards

is

completely

unknown

-LRB-

the

ambiguous

deck

-RRB-

A

bet

on

a

color

pays

a

fixed

sum

-LRB-

e.g.

-RRB-

if

a

card

with

the

chosen

color

is

drawn

and

zero

otherwise

-LRB-

Fig.

-RRB-

Division

of

Humanities

and

Social

Sciences

California

Institute

of

Technology

Pasadena

CA

USA

of

Iowa

Medical

School

Iowa

City

IA

USA

To

whom

correspondence

should

be

addressed

E-mail

camerer@hss.caltech.edu

DECEMBER

Downloaded

from

on

May

REPORTS

A

Card-Deck

B

Knowledge

The

high

temperature

in

Dushanbe

Tajikistan

on

November

is

above

Fahrenheit

C

Informed

Opponent

Your

opponent

will

draw

Ambiguity

Or

Or

Or

Yes

No

Your

opponent

will

draw

Or

Yes

No

The

high

temperature

in

New

York

City

NY

on

November

is

above

Fahrenheit

Or

Or

Fig.

Sample

screens

from

the

experiment

The

conditions

in

the

top

panel

are

called

ambiguous

because

the

subject

is

missing

relevant

information

that

is

available

in

the

risk

conditions

-LRB-

bottom

panel

-RRB-

Subjects

always

choose

between

betting

on

one

of

the

two

options

on

the

left

side

or

taking

the

certain

payoff

on

the

right

-LRB-

A

-RRB-

Card-Deck

treatment

Ambiguity

is

not

knowing

the

exact

proportion

risk

is

knowing

the

number

of

cards

-LRB-

indicated

by

numbers

above

each

deck

-RRB-

-LRB-

B

-RRB-

Knowledge

treatment

Ambiguity

is

knowing

less

about

the

uncertain

events

-LRB-

e.g.

Tajikistan

-RRB-

relative

to

risk

-LRB-

e.g.

New

York

City

-RRB-

-LRB-

C

-RRB-

Informed

Opponent

treatment

Ambiguity

is

betting

against

an

opponent

who

has

more

information

-LRB-

who

drew

a

three-card

sample

from

the

deck

-RRB-

than

in

risk

-LRB-

where

the

opponent

drew

no

cards

from

the

deck

-RRB-

Bets

win

if

subject

chooses

the

realized

color

and

opponent

chooses

the

opposite

color

otherwise

both

take

the

certain

payoff

-LSB-

see

-LRB-

-RRB-

-RSB-

A

Amyg

C

L

Amygdala

R

Amygdala

B

LOFC

y

onset

mean

decision

time

-LRB-

sec

-RRB-

L

OFC

R

OFC

R

y

Ambiguity

T

Risk

Fig.

Regions

showing

greater

activation

in

response

to

ambiguity

than

in

response

to

risk

Randomeffects

analysis

of

all

three

treatments

revealed

regions

that

are

differentially

activated

in

decisionmaking

under

ambiguity

relative

to

risk

-LRB-

P

e

uncorrected

cluster

size

k

Q

voxels

-RRB-

These

regions

include

-LRB-

A

-RRB-

left

amygdala

and

right

amygdala/parahippocampal

gyrus

-LRB-

coronal

section

shown

at

y

in

MNI

space

heat

map

represents

t

statistic

with

degrees

of

freedom

-RRB-

and

-LRB-

B

-RRB-

bilateral

OFC

-LRB-

C

-RRB-

Mean

time

courses

of

amygdala

and

OFC

-LRB-

time

synched

to

trial

onset

dashed

vertical

lines

are

mean

decision

times

error

bars

are

SEM

n

-RRB-

In

experiments

with

these

choices

many

would

rather

bet

on

a

red

draw

from

the

risky

deck

than

on

a

red

draw

from

the

ambiguous

deck

and

similarly

for

blue

-LRB-

-RRB-

If

betting

preferences

are

determined

only

by

probabilities

and

associated

payoffs

this

pattern

is

a

SCIENCE

VOL

paradox

In

theory

disliking

the

bet

on

a

red

draw

from

the

ambiguous

deck

implies

that

its

subjective

probability

is

lower

EPamb

-LRB-

red

-RRB-

G

Prisk

-LRB-

red

-RRB-

The

same

aversion

for

the

blue

bets

implies

Pamb

-LRB-

blue

-RRB-

G

Prisk

-LRB-

blue

-RRB-

But

these

inequalities

and

the

fact

that

the

probabilities

of

red

and

blue

must

sum

to

for

each

deck

imply

Pamb

-LRB-

red

-RRB-

Pamb

-LRB-

blue

-RRB-

G

Prisk

-LRB-

red

-RRB-

Prisk

-LRB-

blue

-RRB-

a

contradiction

The

paradox

can

be

resolved

by

allowing

choices

to

depend

both

on

subjective

probabilities

of

events

and

on

the

ambiguity

of

those

events

-LRB-

-RRB-

More

generally

choices

can

depend

on

how

much

relevant

information

is

missing

or

how

ignorant

people

feel

compared

to

others

-LRB-

-RRB-

We

explored

the

neural

differences

with

varying

levels

of

uncertainty

by

using

a

combination

of

data

from

functional

magnetic

resonance

imaging

-LRB-

fMRI

-RRB-

and

behavioral

data

from

lesion

patients

This

study

builds

on

previous

findings

in

neuroscience

on

reward

and

uncertainty

In

particular

we

focus

on

the

striatum

which

has

been

implicated

in

reward

anticipation

-LRB-

-RRB-

the

orbitofrontal

cortex

-LRB-

OFC

-RRB-

where

patients

with

lesions

perform

poorly

on

behavioral

tasks

involving

uncertainty

such

as

the

Iowa

gambling

task

-LRB-

-RRB-

and

the

amygdala

which

responds

to

ambiguous

facial

cues

and

has

been

hypothesized

as

a

generalized

vigilance

module

in

the

brain

-LRB-

-RRB-

The

fMRI

study

used

three

experimental

treatments

The

Card-Deck

treatment

is

a

baseline

pitting

pure

risk

-LRB-

where

probabilities

are

known

with

certainty

-RRB-

against

pure

ambiguity

The

Knowledge

treatment

uses

choices

about

events

and

facts

which

fall

along

a

spectrum

from

risk

to

ambiguity

In

the

Informed

Opponent

treatment

the

subject

bets

against

another

person

who

has

seen

a

sample

of

cards

from

the

deck

This

opponent

is

therefore

better

informed

about

the

contents

of

the

ambiguous

deck

-LRB-

-RRB-

This

condition

corresponds

to

a

commonly

posited

theory

of

ambiguity

aversion

Even

when

there

is

no

informed

opponent

people

act

as

if

there

is

-LRB-

-RRB-

All

three

treatments

have

one

condition

where

the

subject

is

missing

information

-LRB-

ambiguity

-RRB-

relative

to

the

other

condition

-LRB-

risk

-RRB-

Subjects

made

choices

in

each

treatment

between

certain

amounts

of

money

and

bets

on

card

decks

or

events

-LRB-

-RRB-

The

amounts

of

the

certain

payoff

and

the

bet

payoff

varied

across

trials

In

the

Card-Deck

and

Informed

Opponent

treatments

the

number

and

proportions

of

cards

also

varied

We

estimated

a

general

linear

model

-LRB-

GLM

-RRB-

using

standard

regression

techniques

-LRB-

-RRB-

Two

primary

regressors

were

used

for

each

treatment

one

for

ambiguity

trials

and

one

for

risky

trials

beginning

at

the

onset

of

the

stimulus

and

ending

at

the

time

of

decision

To

find

regions

differentially

activated

by

ambiguity

and

risk

we

performed

a

random-effects

analysis

pooling

all

three

treatments

correcting

for

nonsphericity

-LRB-

-RRB-

Regions

that

were

more

active

during

the

ambiguous

condition

relative

to

the

risk

condi

Risk

DECEMBER

Downloaded

from

on

May

signal

change

REPORTS

tion

included

the

OFC

and

amygdala

-LRB-

Fig.

-RRB-

and

the

dorsomedial

prefrontal

cortex

-LRB-

DMPFC

-RRB-

-LRB-

fig

and

table

-RRB-

These

areas

have

been

implicated

in

integration

of

emotional

and

cognitive

input

-LRB-

OFC

-RRB-

-LRB-

-RRB-

reaction

to

emotional

information

-LRB-

amygdala

-RRB-

-LRB-

-RRB-

and

modulation

of

amygdala

activity

-LRB-

DMPFC

-RRB-

-LRB-

-RRB-

Areas

activated

during

the

risk

condition

relative

to

ambiguity

include

the

dorsal

striatum

-LRB-

caudate

nucleus

-RRB-

-LRB-

Fig.

-RRB-

Furthermore

the

dorsal

striatal

activations

were

also

correlated

with

the

expected

value

of

actual

choices

-LRB-

Fig.

-RRB-

whereas

no

such

correlation

was

observed

in

the

OFC

or

amygdala

-LRB-

tables

and

-RRB-

This

together

with

other

studies

implicating

the

dorsal

striatum

in

reward

prediction

-LRB-

-RRB-

supports

the

hypothesis

that

ambiguity

lowers

the

anticipated

reward

of

decisions

Time

courses

showed

different

patterns

of

activation

in

the

ambiguity

risk

and

risk

ambiguity

regions

Whereas

the

amygdala

and

OFC

reacted

rapidly

at

the

onset

of

the

trial

-LRB-

Fig.

-RRB-

the

dorsal

striatum

activity

built

more

slowly

-LRB-

Fig.

-RRB-

-LRB-

fig

-RRB-

and

peaked

significantly

later

-LRB-

fig

-RRB-

than

those

of

the

amygdala

and

OFC

This

difference

was

present

in

all

three

experimental

treatments

-LRB-

figs

and

-RRB-

and

appeared

to

be

independent

of

subjects

choices

-LRB-

fig

-RRB-

-LRB-

-RRB-

The

temporal

difference

between

these

ambiguity

and

risk

regions

is

consistent

with

the

presence

of

two

interacting

systems

a

Bvigilance

-LSB-

evaluation

system

in

the

amygdala

-LRB-

-RRB-

and

OFC

which

responds

more

rapidly

to

the

stimuli

and

grades

uncertainty

and

a

reward-anticipation

system

in

the

striatum

that

is

further

downstream

Parameters

measuring

ambiguity

and

risk

aversion

-LRB-

g

and

r

respectively

-RRB-

were

estimated

from

a

nonlinear

stochastic

model

of

the

subjects

choice

behavior

in

our

tasks

-LRB-

-RRB-

Ambiguity

aversion

measured

by

g

was

positively

correlated

with

contrast

values

between

ambiguity

and

risk

-LRB-

averaged

over

the

three

treatments

-RRB-

in

the

right

OFC

-LRB-

r

P

G

two-tailed

-RRB-

and

more

weakly

in

the

left

OFC

-LRB-

r

P

G

two-tailed

-RRB-

-LRB-

-RRB-

To

validate

the

fMRI

results

and

establish

that

the

OFC

plays

a

necessary

role

in

distinguishing

levels

of

uncertainty

we

conducted

behavioral

experiments

similar

to

the

card-deck

task

above

using

a

lesion

method

-LRB-

-RRB-

Twelve

neurological

subjects

with

focal

brain

lesions

were

partitioned

into

two

groups

those

whose

lesions

included

the

most

significant

activation

focus

in

the

OFC

revealed

in

our

fMRI

study

-LRB-

n

-RRB-

and

a

comparison

group

-LRB-

temporal

lobe

damage

patients

-RRB-

whose

lesions

did

not

overlap

with

any

of

our

fMRI

foci

-LRB-

n

-RRB-

The

two

groups

had

similar

etiology

IQ

mathematical

ability

and

performance

on

other

background

tasks

-LRB-

table

-RRB-

Two-dimensional

confidence

interval

analysis

-LRB-

Fig.

-RRB-

showed

that

frontal

patients

are

risk

and

ambiguity-neutral

-LRB-

i.e.

the

hypothesis

that

g

r

can

not

be

rejected

-RRB-

This

differed

from

the

comparison

group

who

ap

A

Striatum

B

L

Striatum

C

Knowledge

Card-Deck

R

signal

change

time

-LRB-

sec

-RRB-

R

Striatum

y

y

Expected

value

-LRB-

Card-Deck

-RRB-

T

onset

mean

decision

Expected

value

-LRB-

Knowledge

-RRB-

Ambiguity

Risk

risk

preference

parameter

Ambiguity

Preferring

Risk

Preferring

Ambiguity

Averse

Risk

Preferring

Ambiguity

Preferring

Ambiguity

Averse

Risk

Averse

Risk

Averse

ambiguity

preference

parameter

Frontal

Estimate

Frontal

bootstrap

estimates

Frontal

bootstrap

ellipse

Comparison

Estimate

Comparison

bootstrap

estimates

Comparison

bootstrap

ellipse

Fig.

Risk

and

ambiguity

attitudes

of

OFC

patients

-LRB-

n

-RRB-

and

lesion

comparisons

-LRB-

n

-RRB-

with

associated

confidence

intervals

The

risk

neutral

line

-LRB-

r

-RRB-

and

the

ambiguity

neutral

line

-LRB-

g

-RRB-

demarcate

four

quadrants

as

labeled

Open

symbols

plot

maximum

likelihood

estimates

of

a

group-level

stochastic

choice

model

Frontals

-LRB-

g

r

-RRB-

lesion

comparisons

-LRB-

g

r

-RRB-

-LSB-

see

-LRB-

-RRB-

-RSB-

Solid

symbols

represent

bootstrapped

-LRB-

g

r

-RRB-

estimates

Ellipses

are

twodimensional

confidence

intervals

around

the

bootstrapped

data

Angle

of

the

ellipse

reflects

correlation

between

r

and

g

-LRB-

for

frontal

for

comparison

-RRB-

peared

to

be

risk

and

ambiguity-averse

The

OFC-lesioned

group

therefore

did

not

distinguish

between

degrees

of

uncertainty

-LRB-

ambiVOL

SCIENCE

guity

and

risk

-RRB-

This

is

behaviorally

abnormal

but

is

consistent

ironically

with

the

logic

of

subjective

expected

utility

theory

DECEMBER

Downloaded

from

on

May

Fig.

Regions

showing

greater

activation

in

response

to

risk

than

in

response

to

ambiguity

Randomeffects

analysis

of

all

three

treatments

revealed

brain

regions

that

are

differentially

activated

in

decision-making

under

risk

These

regions

include

-LRB-

A

-RRB-

dorsal

striatum

as

well

as

precuneus

and

premotor

cortex

-LRB-

table

-RRB-

-LRB-

P

e

uncorrected

cluster

size

k

Q

voxels

-RRB-

-LRB-

B

-RRB-

Mean

time

courses

for

risk

regions

-LRB-

time

synched

to

trial

onset

dashed

vertical

lines

are

mean

decision

times

error

bars

are

SEM

n

-RRB-

-LRB-

C

-RRB-

Regions

of

the

dorsal

striatum

significantly

correlated

with

expected

values

of

subjects

choices

in

risk

condition

of

Card-Deck

treatment

-LRB-

red

-RRB-

and

both

risk

and

ambiguity

conditions

of

Knowledge

treatment

-LRB-

blue

-RRB-

-LRB-

P

G

uncorrected

cluster

size

k

Q

voxels

-RRB-

REPORTS

Together

with

the

fMRI

results

these

data

suggest

a

neural

system

for

evaluating

general

uncertainty

Both

the

amygdala

and

OFC

are

known

to

receive

rapid

multimodal

sensory

input

both

are

bidirectionally

connected

and

are

known

to

function

together

in

evaluating

the

value

of

stimuli

-LRB-

-RRB-

and

both

are

likely

involved

in

detecting

salient

and

relevant

stimuli

of

uncertain

value

The

latter

function

has

been

hypothesized

especially

for

the

amygdala

-LRB-

-RRB-

Such

a

function

also

provides

a

reward-related

signal

that

can

motivate

behavior

by

virtue

of

the

known

connections

between

the

amygdala/OFC

and

the

striatum

-LRB-

-RRB-

Although

the

circuit

is

assessed

here

in

the

context

of

a

neuroeconomic

experiment

we

believe

that

it

subserves

general

aspects

of

how

organisms

explore

their

environment

Under

uncertainty

the

brain

is

alerted

to

the

fact

that

information

is

missing

that

choices

based

on

the

information

available

therefore

carry

more

unknown

-LRB-

and

potentially

dangerous

-RRB-

consequences

and

that

cognitive

and

behavioral

resources

must

be

mobilized

in

order

to

seek

out

additional

information

from

the

environment

Understanding

the

neural

basis

of

choice

under

uncertainty

is

important

because

it

is

a

fundamental

activity

at

every

societal

level

with

examples

as

diverse

as

people

saving

for

retirement

companies

pricing

insurance

and

countries

evaluating

military

social

and

environmental

risks

-LRB-

-RRB-

The

choices

can

vary

greatly

in

the

level

of

information

available

to

the

decisionmaker

about

outcome

probabilities

Standard

decision

theory

however

precludes

agents

from

acting

differently

in

the

face

of

risk

and

ambiguity

Our

results

show

that

this

hypothesis

is

wrong

on

both

the

behavioral

and

neural

level

and

suggest

a

unified

treatment

of

ambiguity

and

risk

as

limiting

cases

of

a

general

system

evaluating

uncertainty

For

neuroscientists

these

results

introduce

the

important

concept

of

varying

degrees

of

uncertainty

that

is

missing

from

previous

studies

of

reward

and

decision-making

More

generally

this

study

shows

the

value

of

combining

ideas

and

tools

from

social

and

biological

sciences

-LRB-

-RRB-

References

and

Notes

C.

Camerer

M.

Weber

J.

Risk

Uncert

-LRB-

-RRB-

D.

Ellsberg

Q.

J.

Econ

-LRB-

-RRB-

S.

Becker

F.

Brownson

J.

Polit

Econ

-LRB-

-RRB-

K.

MacCrimmon

Risk

and

Uncertainty

K.

Borch

J.

Mossin

Eds

-LRB-

Macmillan

London

-RRB-

If

ambiguous

probabilities

are

subadditive

then

Pamb

-LRB-

red

-RRB-

Pamb

-LRB-

blue

-RRB-

represents

reserved

belief

and

indexes

the

degree

of

aversion

to

ambiguity

Some

models

assume

that

probabilities

are

additive

but

are

set-valued

and

assume

that

the

worst

probability

in

the

set

determines

their

chances

-LRB-

-RRB-

This

model

and

others

are

silent

about

possible

neural

circuitry

D.

Schmeidler

Econometrica

-LRB-

-RRB-

I.

Gilboa

D.

Schmeidler

J.

Math

Econ

-LRB-

-RRB-

D.

Frisch

J.

Baron

J.

Behav

Decision

Making

-LRB-

-RRB-

C.

Fox

A.

Tversky

Q.

J.

Econ

-LRB-

-RRB-

W.

Schultz

Nat

Rev.

Neurosci

-LRB-

-RRB-

A.

Bechara

D.

Tranel

H.

Damasio

Brain

-LRB-

-RRB-

H.

Kim

et

al.

J.

Cogn

Neurosci

-LRB-

-RRB-

H.

Kim

L.

H.

Somerville

T.

Johnstone

A.

L.

Alexander

P.

J.

Whalen

Neuroreport

-LRB-

-RRB-

E.

Phelps

et

al.

J.

Cogn

Neurosci

-LRB-

-RRB-

The

Informed

Opponent

treatment

is

as

follows

-LRB-

i

-RRB-

Both

subjects

choose

a

color

with

the

opponent

having

sampled

the

specified

number

of

cards

from

the

ambiguous

deck

-LRB-

ii

-RRB-

a

card

is

chosen

from

the

deck

and

-LRB-

iii

-RRB-

the

person

with

the

color

that

matches

that

of

the

card

chosen

wins

the

bet

Mismatches

make

nothing

-LRB-

-RRB-

The

Informed

Opponent

hypothesis

is

that

bets

on

ambiguous

card

decks

and

lowknowledge

events

although

normatively

different

from

bets

against

the

informed

opponent

are

generated

by

similar

neural

circuitry

Similarity

of

time

courses

in

the

amygdala

OFC

and

striatum

across

all

three

treatments

-LRB-

figs

and

-RRB-

is

consistent

with

this

hypothesis

A.

Kuhberger

J.

Perner

J.

Behav

Decision

Making

-LRB-

-RRB-

See

supporting

data

on

Science

Online

H.

Critchley

C.

Mathias

R.

Dolan

Neuron

-LRB-

-RRB-

A.

Bechara

H.

Damasio

A.

Damasio

Ann

N.Y.

Acad

Sci

-LRB-

-RRB-

R.

Adolphs

Curr

Opin

Neurobiol

-LRB-

-RRB-

H.

Critchley

R.

Elliot

C.

Mathias

R.

Dolan

J.

Neurosci

-LRB-

-RRB-

J.

O'Doherty

et

al.

Science

-LRB-

-RRB-

B.

Knutson

C.

Adams

G.

Fong

D.

Hommer

J.

Neurosci

-LRB-

-RRB-

One

earlier

positron

emission

tomography

study

-LRB-

-RRB-

also

found

differential

activation

in

the

caudate

during

risk

relative

to

ambiguity

-LRB-

C-CAll.pdf

-RRB-

The

areas

that

are

differentially

activated

by

the

choice

that

subjects

make

-LRB-

gamble

or

certain

payoff

-RRB-

include

the

caudate

head

and

the

insula

-LRB-

table

-RRB-

which

are

also

not

significantly

interacting

with

ambiguity/risk

P.

J.

Whalen

Curr

Direct

Psychol

Sci

-LRB-

-RRB-

Aversion

to

risk

is

measured

by

the

concavity

of

the

subjective

utility

function

for

money

u

-LRB-

x

-RRB-

xr

Lower

r

corresponds

to

more

risk

aversion

Aversion

to

ambiguity

-LRB-

controlling

for

risk

aversion

-RRB-

is

measured

by

the

degree

to

which

probabilities

P

are

underweighted

when

they

are

ambiguous

w

-LRB-

P

-RRB-

Pg

Higher

g

corresponds

to

more

ambiguity

aversion

Best

fitting

values

of

r

and

g

are

estimated

via

maximum

likelihood

-LRB-

-RRB-

D.

Gaffan

E.

A.

Murray

M.

Fabre-Thorpe

Eur

J.

Neurosci

-LRB-

-RRB-

R.

B.

Adams

Jr.

H.

L.

Gordon

A.

A.

Baird

N.

Ambady

R.

E.

Kleck

Science

-LRB-

-RRB-

D.

Amaral

J.

Price

A.

Pitkanen

S.

Carmichael

in

The

Amygdala

Neurobiological

Aspects

of

Emotion

Memory

and

Mental

Dysfunction

J.

P.

Aggleton

Ed

-LRB-

Wiley

New

York

-RRB-

pp.

P.

Glimcher

A.

Rustichini

Science

-LRB-

-RRB-

S.

M.

McClure

D.

Laibson

G.

Loewenstein

J.

D.

Cohen

Science

-LRB-

-RRB-

A.

Rustichini

J.

Dickhaut

P.

Ghiradato

K.

Smith

J.

Pardo

Games

Econ

Behav

-LRB-

-RRB-

We

thank

K.

Scheer

and

M.

Koenigs

for

data

collection

and

P.

Bossaerts

and

S.

Quartz

for

valuable

input

in

the

planning

stages

of

this

work

Supported

by

NSF

grant

SES

-LRB-

C.C.

and

R.A.

-RRB-

the

MacArthur

Foundation

Preferences

Network

-LRB-

C.C.

-RRB-

Caltech

grant

-LRB-

C.C.

-RRB-

NIH

grants

-LRB-

R.A.

-RRB-

and

-LRB-

D.T.

-RRB-

and

the

David

and

Lucile

Packard

Foundation

Supporting

Online

Material

Materials

and

Methods

Figs

to

Tables

to

References

May

accepted

October

science

A

Conserved

Checkpoint

Monitors

Meiotic

Chromosome

Synapsis

in

Caenorhabditis

elegans

Needhi

and

Abby

F.

We

report

the

discovery

of

a

checkpoint

that

monitors

synapsis

between

homologous

chromosomes

to

ensure

accurate

meiotic

segregation

Oocytes

containing

unsynapsed

chromosomes

selectively

undergo

apoptosis

even

if

a

germline

DNA

damage

checkpoint

is

inactivated

This

culling

mechanism

is

specifically

activated

by

unsynapsed

pairing

centers

cis-acting

chromosome

sites

that

are

also

required

to

promote

synapsis

in

Caenorhabditis

elegans

Apoptosis

due

to

synaptic

failure

also

requires

the

C.

elegans

homolog

of

a

budding

yeast

pachytene

checkpoint

gene

which

suggests

that

this

surveillance

mechanism

is

widely

conserved

Meiosis

requires

two

successive

cell

divisions

one

in

which

homologous

chromosomes

separate

and

a

second

that

partitions

sister

chromatids

Accurate

segregation

depends

on

the

establishment

of

physical

linkages

-LRB-

chiasmata

-RRB-

between

homologous

chromosomes

during

meiotic

prophase

Chromosome

pairing

the

polymerization

of

the

synaptonemal

complex

between

paired

homologs

-LRB-

synapsis

-RRB-

and

crossover

recombination

are

all

required

to

generate

chiasmata

Life

Sciences

Division

Lawrence

Berkeley

National

Laboratory

of

Molecular

and

Cell

Biology

University

of

California

Berkeley

Berkeley

CA

USA

To

whom

correspondence

should

be

addressed

E-mail

afdernburg@lbl.gov

which

enable

proper

chromosome

alignment

on

the

meiotic

spindle

Defects

in

these

early

meiotic

events

can

lead

to

cell

cycle

arrest

or

apoptosis

indicating

that

the

events

are

monitored

by

checkpoints

In

budding

yeast

a

Bpachytene

checkpoint

-LSB-

responds

to

defects

in

homolog

synapsis

and/or

recombination

Ereviewed

in

-LRB-

-RRB-

Mammalian

meiosis

may

have

two

distinct

checkpoints

one

that

responds

to

synaptic

failure

and

one

that

responds

to

DNA

damage

-LRB-

-RRB-

Because

synapsis

and

recombination

are

obligately

coupled

in

both

Saccharomyces

cerevisiae

-LRB-

-RRB-

and

mice

-LRB-

-RRB-

it

has

been

ambiguous

whether

these

checkpoints

are

triggered

by

recombination

defects

or

asynapsis

Here

we

have

exploited

the

knowledge

that

synapsis

can

be

complete

SCIENCE

VOL

DECEMBER

Downloaded

from

on

May

O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O B O O O O O O O O O O O O O O B O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O B O B O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O B O O O O O O O B O B O B O B O B O O O O O B O B O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ambiguity
decision making
neuroscience
uncertainty
amygdala
risk


letters

to

nature

Kiss

I.

Z.

Zhai

Y.

Hudson

J.

L.

Emerging

coherence

in

a

population

of

chemical

oscillators

Science

-LRB-

-RRB-

Levine

J.

D.

Funes

P.

Dowse

H.

B.

Hall

J.

C.

Resetting

the

circadian

clock

by

social

experience

in

Drosophila

melanogaster

Science

-LRB-

-RRB-

Winfree

A.

T

The

Geometry

of

Biological

Time

edn

-LRB-

Springer

New

York

-RRB-

Goldbeter

A.

Computational

approaches

to

cellular

rhythms

Nature

-LRB-

-RRB-

Gonze

D.

Halloy

J.

Goldbeter

A.

Robustness

of

circadian

rhythms

with

respect

to

molecular

noise

Proc

Natl

Acad

Sci

USA

-LRB-

-RRB-

Supplementary

Information

accompanies

the

paper

on

Acknowledgements

We

thank

S.

Golden

for

the

strain

and

for

advice

D.

Peoples

for

advice

and

technical

assistance

B.

Houchmandzadeh

J.

Paulsson

J.

Vilar

C.

Weitz

and

M.

Young

for

discussions

and

N.

Questembert-Balaban

E.

Kussell

and

M.

Vallade

for

comments

on

the

manuscript

This

work

was

supported

partially

by

Princeton

University

through

the

Lewis

Thomas

Fellowship

-LRB-

I.M.

-RRB-

the

National

Institutes

of

Health

the

Howard

Hughes

Medical

Institute

and

the

Centre

National

de

Recherche

Scientifique

through

an

ATIP

and

an

AC

Dynamique

et

reactivite

des

assemblages

biologiques

Competing

interests

statement

The

authors

declare

that

they

have

no

competing

financial

interests

Correspondence

and

requests

for

materials

should

be

addressed

to

I.M.

-LRB-

imihalce@spectro.ujf-grenoble.fr

-RRB-

Evolutionary

changes

in

cis

and

trans

gene

regulation

Patricia

J.

Wittkopp

Belinda

K.

Haerum

Andrew

G.

Clark

Department

of

Molecular

Biology

and

Genetics

Cornell

University

Ithaca

New

York

USA

hybrids

will

be

different

from

that

in

one

or

both

of

the

parental

species

As

a

result

the

relative

allelic

expression

in

hybrids

will

differ

from

the

relative

gene

expression

between

species

Transregulatory

divergence

was

inferred

for

any

gene

with

significant

differences

in

the

ratio

of

species-specific

transcripts

between

hybrids

and

the

parental

species

To

measure

the

relative

abundances

of

species-specific

transcripts

we

analysed

RNA

and

DNA

extracted

separately

from

pools

of

female

flies

containing

either

hybrids

or

D.

melanogaster

and

D.

simulans

individuals

-LRB-

Fig.

-RRB-

Four

complementary

DNA

samples

were

generated

from

each

RNA

extraction

and

used

to

measure

the

relative

abundance

of

species-specific

transcripts

At

least

two

replicate

hybrid

pools

-LRB-

eight

cDNA

measurements

in

total

-RRB-

and

four

replicate

parental

pools

-LRB-

cDNA

measurements

in

total

-RRB-

were

analysed

for

each

gene

The

ratio

of

D.

melanogaster

to

D.

simulans

alleles

was

also

measured

in

genomic

DNA

from

each

pool

in

duplicate

and

used

to

correct

cDNA

ratios

for

allelic

differences

in

extraction

and/or

amplification

-LRB-

Supplementary

Information

-RRB-

For

each

gene

the

ratio

of

expression

between

species

-LRB-

designated

Mel/Sim

-RRB-

and

the

ratio

of

species-specific

transcripts

in

hybrids

-LRB-

designated

-RRB-

were

quantified

normalized

and

averaged

across

replicate

pools

was

used

to

measure

the

relative

abundance

of

D.

melanogaster

and

D.

simulans

alleles

directly

in

cDNA

and

DNA

samples

A

single

nucleotide

difference

that

distinguished

the

species-specific

transcripts

was

identified

for

each

gene

and

polymerase

chain

reaction

-LRB-

PCR

-RRB-

primers

were

used

to

amplify

a

region

of

DNA

containing

the

divergent

site

-LRB-

Fig.

-RRB-

After

denaturing

the

PCR

products

an

internal

primer

-LRB-

Pyro-reverse

in

Fig.

-RRB-

was

annealed

and

extended

one

base

at

a

time

in

a

Pyrosequencing

Differences

in

gene

expression

are

central

to

evolution

Such

differences

can

arise

from

cis-regulatory

changes

that

affect

transcription

initiation

transcription

rate

and/or

transcript

stability

in

an

allele-specific

manner

or

from

trans-regulatory

changes

that

modify

the

activity

or

expression

of

factors

that

interact

with

cis-regulatory

Both

cis

and

transregulatory

changes

contribute

to

divergent

gene

expression

but

their

respective

contributions

remain

largely

Here

we

examine

the

distribution

of

cis

and

trans-regulatory

changes

underlying

expression

differences

between

closely

related

Drosophila

species

D.

melanogaster

and

D.

simulans

and

show

functional

cis-regulatory

differences

by

comparing

the

relative

abundance

of

species-specific

transcripts

in

Differences

in

trans-regulatory

activity

were

inferred

by

comparing

the

ratio

of

allelic

expression

in

hybrids

with

the

ratio

of

gene

expression

between

species

Of

genes

with

interspecific

expression

differences

had

differences

in

cis-regulation

and

these

changes

were

sufficient

to

explain

expression

divergence

for

about

half

of

the

genes

Trans-regulatory

differences

affected

-LRB-

of

-RRB-

of

genes

and

were

always

accompanied

by

cisregulatory

changes

These

data

indicate

that

interspecific

expression

differences

are

not

caused

by

select

trans-regulatory

changes

with

widespread

effects

but

rather

by

many

cis-acting

changes

spread

throughout

the

genome

D.

melanogaster

and

D.

simulans

diverged

about

Myr

yet

are

still

able

to

mate

and

produce

viable

-LRB-

but

sterile

-RRB-

offspring

Their

compatibility

allowed

us

to

identify

cis

and

trans-regulatory

changes

by

comparing

the

regulation

of

species-specific

alleles

in

a

common

hybrid

genetic

background

Differential

expression

of

two

alleles

in

the

same

cellular

environment

indicates

functional

cisregulatory

thus

asymmetric

allelic

expression

in

hybrids

implies

cis-regulatory

divergence

If

trans-regulation

diverges

between

species

the

collection

of

trans-acting

factors

in

NATURE

VOL

JULY

CGAGATCGGCGGCACCAAGCagaagctgaagatcctgcaccc

g

g

a

a

a

AAGAGTGAGCACAACCAcaaccacggactggtgctcgaaAACCACAGCGAGTCGCACCACG

Figure

measures

allelic

gene

expression

a

Overview

of

sample

collection

b

Example

assay

of

Aligned

coding

sequences

from

D.

melanogaster

-LRB-

-RRB-

and

D.

simulans

-LRB-

-RRB-

identify

transcribed

fixed

differences

-LRB-

box

-RRB-

The

location

of

PCR

and

Pyrosequencing

primers

are

shown

with

an

asterisk

indicating

the

biotinylated

PCR

primer

c

Pyrosequencing

produces

a

pyrogram

with

peaks

whose

height

is

directly

proportional

to

the

quantity

of

nucleotide

added

to

the

extending

Pyrosequencing

primer

Pyrograms

from

reactions

using

genomic

DNA

from

D.

melanogaster

-LRB-

left

-RRB-

D.

simulans

-LRB-

middle

-RRB-

and

both

species

-LRB-

right

-RRB-

are

shown

Arrows

indicate

peaks

reflecting

nucleotide

incorporation

at

the

divergent

site

ES

indicates

the

addition

of

enzymes

and

substrate

respectively

Nature

Publishing

Group

letters

to

nature

reaction

which

couples

DNA

synthesis

with

a

series

of

enzymatic

reactions

that

produce

stoichiometric

quantities

of

The

amount

of

light

generated

by

the

incorporation

of

species-specific

bases

was

directly

proportional

to

the

relative

abundance

of

the

D.

melanogaster

and

D.

simulans

alleles

in

the

PCR

template

-LRB-

Fig.

Supplementary

Fig.

-RRB-

Thirty-four

genes

previously

shown

to

have

a

significant

expression

difference

between

D.

melanogaster

and

D.

simulans

were

selected

for

of

these

genes

showed

significant

expression

differences

between

species

in

this

study

ranging

from

to

-LRB-

Fig.

t-test

Mel/Sim

P

-RRB-

Because

we

examined

a

different

developmental

stage

in

different

strains

of

flies

from

those

in

the

initial

it

is

not

surprising

that

we

observed

distinct

expression

levels

Comparison

with

an

independent

microarray

study

of

expression

differences

between

D.

melanogaster

and

D.

simulans

indicates

that

this

gene

set

is

representative

of

interspecific

expression

differences

genomewide

-LRB-

Supplementary

Information

-RRB-

Unequal

expression

of

the

D.

melanogaster

and

D.

simulans

alleles

in

a

hybrid

genetic

background

indicates

differences

in

cis-regulation

Allelic

expression

of

species-specific

alleles

in

hybrids

showed

that

nearly

all

-LRB-

of

-RRB-

genes

with

interspecific

expression

differences

had

evolved

changes

in

cis-regulatory

function

-LRB-

Fig.

t-test

P

-RRB-

Differential

expression

of

species-specific

alleles

ranged

from

to

with

the

more

highly

expressed

allele

derived

from

D.

melanogaster

half

of

the

time

Two

of

the

five

genes

with

similar

expression

between

species

also

showed

evidence

of

cis-regulatory

divergence

-LRB-

Fig.

b

arrows

t-test

P

-RRB-

indicating

that

compensatory

cis

and

trans-regulatory

changes

have

evolved

that

maintain

gene

expression

To

ensure

that

differences

in

allelic

expression

were

caused

by

cis-regulatory

changes

rather

than

parent-of-origin

effects

we

compared

the

ratio

of

species-specific

transcripts

for

genes

in

hybrids

from

reciprocal

crosses

The

direction

of

cross

used

to

produce

the

hybrids

generally

had

a

negligible

effect

on

the

expression

levels

of

the

D.

melanogaster

and

D.

simulans

alleles

-LRB-

Supplementary

Fig.

-RRB-

Cis-regulatory

differences

seem

to

be

extremely

common

between

orthologous

genes

in

D.

melanogaster

and

D.

simulans

But

are

changes

in

cis-regulatory

function

usually

the

primary

cause

of

interspecific

expression

differences

or

do

they

contribute

relatively

little

in

comparison

with

genetic

changes

affecting

transregulation

To

address

this

question

we

compared

the

expression

difference

between

species

-LRB-

Mel/Sim

-RRB-

with

the

relative

abundance

of

species-specific

transcripts

in

hybrids

-LRB-

Fig.

-RRB-

If

cis-regulatory

divergence

completely

explains

the

difference

between

species

the

hybrid

and

parental

expression

ratios

will

be

the

same

-LRB-

Mel/Sim

-RRB-

and

points

in

Fig.

will

fall

on

the

diagonal

In

contrast

if

only

trans-regulatory

differences

cause

the

divergent

expression

the

D.

melanogaster

and

D.

simulans

alleles

will

be

equally

expressed

in

hybrids

-LRB-

-RRB-

and

Figure

Cis-regulatory

changes

frequently

contribute

to

interspecific

expression

differences

a

Relative

gene

expression

levels

between

species

-LRB-

Mel/Sim

-RRB-

b

Relative

allelic

expression

in

hybrids

-LRB-

-RRB-

The

gene

order

is

the

same

in

both

panels

with

data

plotted

on

a

logarithmic

scale

The

normalized

ratio

of

D.

melanogaster

to

D.

simulans

transcripts

is

shown

at

the

left

the

corresponding

fold

expression

changes

are

shown

at

the

right

Error

bars

show

confidence

intervals

Arrows

indicate

genes

with

compensatory

cis

and

trans-regulatory

changes

and

arrowheads

show

genes

with

conserved

cis

and

trans-regulation

Asterisks

indicate

the

only

gene

that

showed

divergent

expression

without

significant

cis-regulatory

differences

Figure

Evolutionary

changes

in

both

cis

and

trans-regulation

underlie

interspecific

expression

differences

The

relative

expression

between

species

-LRB-

Mel/Sim

-RRB-

is

plotted

against

the

relative

expression

of

species-specific

alleles

in

hybrids

-LRB-

-RRB-

on

a

logarithmic

scale

The

dashed

box

indicates

twofold

expression

differences

Error

bars

show

confidence

intervals

Genes

are

colour-coded

according

to

our

inference

of

cis

and

trans-regulatory

changes

red

cis-regulatory

changes

can

completely

explain

interspecific

expression

differences

blue

cis-regulatory

changes

account

for

a

fraction

of

the

expression

difference

with

trans-regulatory

changes

explaining

the

remaining

fraction

green

cis

and

trans-regulatory

changes

with

antagonistic

effects

have

evolved

yellow

which

displayed

no

evidence

of

cis

or

trans-regulatory

divergence

-LRB-

P

and

P

respectively

-RRB-

despite

a

small

significant

expression

difference

between

species

-LRB-

Mel/Sim

P

-RRB-

The

diagonal

line

indicates

cisregulatory

divergence

the

bold

horizontal

line

indicates

trans-regulatory

divergence

NATURE

VOL

JULY

Nature

Publishing

Group

letters

to

nature

points

will

fall

along

the

horizontal

axis

Genes

affected

by

both

cisand

trans-regulatory

divergence

are

indicated

in

Fig.

by

points

that

do

not

fall

on

either

of

these

lines

Cis-regulatory

changes

could

completely

explain

interspecific

expression

differences

for

almost

half

-LRB-

of

-RRB-

of

the

genes

with

divergent

cis-regulatory

functions

-LRB-

red

in

Fig.

t-test

Mel

Sim

P

-RRB-

The

remaining

genes

displayed

evidence

of

both

cis

and

trans-regulatory

changes

-LRB-

Mel

and

Mel/Sim

-RRB-

For

eight

genes

differences

in

cis-regulation

were

less

extreme

than

the

interspecific

expression

difference

-LRB-

blue

in

Fig.

-RRB-

Genetic

changes

affecting

trans-acting

factors

are

responsible

for

the

remaining

expression

difference

between

species

The

other

eight

genes

showed

allelic

expression

differences

in

hybrids

that

were

greater

than

or

in

the

opposite

direction

from

the

interspecific

differences

-LRB-

green

in

Fig.

-RRB-

Evolutionary

changes

in

cis

and

trans-regulation

with

opposing

effects

on

gene

expression

must

underlie

interspecific

differences

for

these

genes

Such

changes

are

consistent

with

the

coevolution

of

cis

and

trans-regulatory

factors

Overall

cis-regulatory

differences

affected

of

genes

with

divergent

expression

between

Drosophila

species

with

at

least

also

influenced

by

trans-regulatory

changes

The

presence

of

trans-regulatory

differences

was

not

related

to

the

magnitude

of

the

expression

difference

between

species

-LRB-

Kruskal

Wallis

test

P

-RRB-

The

distribution

of

cis

and

trans-regulatory

changes

provides

insight

into

the

evolution

of

regulatory

networks

To

a

first

approximation

these

networks

are

composed

of

two

classes

of

regulatory

-LRB-

for

example

those

encoding

transcription

factors

or

signalling

molecules

-RRB-

and

structural

-LRB-

for

example

those

encoding

enzymes

or

cellular

components

-RRB-

Regulatory

genes

-LRB-

Fig.

filled

circles

-RRB-

comprise

internal

connections

in

the

network

and

changes

in

their

expression

often

have

trans-acting

effects

on

the

expression

of

many

other

genes

In

contrast

structural

genes

-LRB-

Fig.

open

circles

-RRB-

typically

lie

at

terminal

nodes

of

the

network

where

changes

in

their

expression

have

more

limited

effects

on

expression

of

other

genes

On

the

basis

of

the

available

annotations

of

molecular

our

data

set

contains

primarily

structural

genes

-LRB-

Supplementary

Table

-RRB-

Analysis

of

structural

genes

provides

a

complete

view

of

the

regulatory

network

with

gene-specific

changes

at

terminal

nodes

in

the

network

detected

as

cis-regulatory

changes

and

genetic

changes

located

in

upstream

regulatory

genes

identifiable

as

trans-regulatory

differences

Two

extreme

models

can

be

considered

to

explain

the

myriad

differences

in

gene

expression

between

species

a

few

cis-regulatory

changes

affecting

regulatory

genes

could

have

widespread

transacting

effects

on

the

expression

of

many

downstream

genes

-LRB-

Fig.

-RRB-

or

each

structural

gene

's

expression

could

be

altered

by

its

own

cisacting

genetic

changes

-LRB-

Fig.

-RRB-

We

found

that

interspecific

expression

differences

were

almost

always

caused

at

least

in

part

by

cis-regulatory

changes

in

structural

genes

and

that

differences

in

trans-regulation

also

affected

half

of

the

genes

-LRB-

Fig.

-RRB-

The

prevalence

of

cis-regulatory

changes

suggests

that

differences

in

gene

expression

between

D.

melanogaster

and

D.

simulans

could

have

evolved

by

changing

the

expression

of

structural

genes

one

gene

at

a

time

However

regulatory

differences

observed

in

extant

flies

might

not

have

been

the

original

source

of

expression

divergence

With

the

availability

of

DNA

microarrays

whole

genomes

can

now

be

compared

for

differences

in

gene

expression

within

and

between

species

By

combining

expression

analysis

with

genetic

methods

such

as

quantitative

trait

locus

mapping

or

the

approach

presented

here

the

genetic

basis

of

variable

gene

expression

is

beginning

to

be

Distinguishing

between

cis

and

trans-regulatory

changes

is

the

first

step

in

this

process

and

it

provides

a

crucial

foothold

for

identifying

the

specific

nucleotide

changes

underlying

gene

expression

differences

A

Methods

Fly

strains

and

crosses

A

D.

melanogaster

zygotic

hybrid

rescue

-LRB-

zhr

-RRB-

strain

-LRB-

provided

by

A.

Orr

-RRB-

and

the

Tsimbazaza

D.

simulans

strain

-LRB-

provided

by

H.

Hollocher

-RRB-

were

crossed

to

produce

hybrids

These

strains

reduce

premating

isolation

between

D.

melanogaster

and

D.

simulans

and

rescue

the

lethality

of

hybrid

Female

flies

from

parental

lines

and

hybrid

females

from

reciprocal

interspecific

crosses

were

collected

within

h

of

emergence

aged

for

h

snap-frozen

in

liquid

nitrogen

and

stored

at

The

D.

melanogaster

In

-LRB-

-RRB-

AB

strain

and

D.

simulans

and

male

hybrid

rescue

-LRB-

mhr

-RRB-

strains

-LRB-

provided

by

D.

Barbash

-RRB-

were

also

used

for

DNA

sequencing

Gene

selection

Genes

were

selected

on

the

basis

of

the

microarray

data

of

Rifkin

et

al.

which

measured

changes

in

gene

expression

during

metamorphosis

between

D.

melanogaster

and

D.

simulans

Each

gene

selected

showed

evidence

of

lineage-specific

selection

and

had

expression

in

D.

simulans

that

was

at

least

twofold

different

from

all

four

strains

of

D.

melanogaster

analysed

Because

morphological

identification

of

hybrid

females

during

the

larval

and

pupal

stages

examined

by

Rifkin

et

al.

is

difficult

we

analysed

adult

female

flies

instead

In

addition

because

the

fly

strains

used

by

Rifkin

et

al.

do

not

mate

we

used

the

zhr

and

Tsimbazaza

strains

Extraction

of

nucleic

acids

and

preparation

of

cDNA

For

each

pool

of

flies

DNA

and

RNA

were

extracted

separately

from

a

single

homogenate

by

using

a

modified

protocol

for

the

SV

Total

RNA

Isolation

System

-LRB-

Promega

-RRB-

The

homogenate

was

passed

over

a

column

that

retained

DNA

the

flowthrough

-LRB-

containing

RNA

and

proteins

-RRB-

was

then

passed

over

a

second

column

that

bound

RNA

RNA

samples

were

treated

with

DNase

during

extraction

and

immediately

before

cDNA

synthesis

RNA

was

reverse

transcribed

into

cDNA

with

the

use

of

a

poly

-LRB-

T

-RRB-

primer

and

standard

protocols

PCR

reactions

with

primers

annealing

to

introns

confirmed

that

cDNA

samples

were

free

of

genomic

DNA

Protocols

are

available

from

the

authors

on

request

Allele

quantification

with

Pyrosequencing

Using

primer

sequences

listed

in

ref

we

amplified

and

sequenced

base

pairs

of

transcribed

DNA

from

each

gene

in

two

D.

melanogaster

and

three

D.

simulans

strains

After

the

identification

of

species-specific

nucleotide

differences

PCR

primers

annealing

to

conserved

sequences

were

used

to

amplify

regions

of

sequence

including

the

divergent

sites

An

internal

primer

also

matching

a

conserved

sequence

was

then

annealed

Pyrosequencing

reactions

performed

in

accordance

with

the

manufacturer

's

instructions

-LRB-

-RRB-

were

used

to

measure

the

relative

abundance

of

the

two

alleles

in

genomic

DNA

and

cDNA

samples

from

both

parental

and

hybrid

pools

Primer

sequences

and

PCR

annealing

temperatures

are

available

from

the

authors

on

request

Pyrosequencing

software

reports

a

peak

height

directly

proportional

to

the

number

of

molecules

incorporated

into

the

growing

DNA

chain

Custom

Perl

scripts

were

used

to

calculate

the

ratio

of

peak

heights

associated

with

incorporation

of

allele-specific

bases

-LRB-

or

Mel/Sim

-RRB-

which

corresponds

to

the

relative

abundance

of

the

D.

melanogaster

and

D.

simulans

alleles

in

the

starting

sample

-LRB-

Supplementary

Fig.

-RRB-

cDNA

ratios

were

normalized

with

genomic

DNA

measurements

as

described

in

Supplementary

Information

Because

both

alleles

are

extracted

and

measured

in

a

single

sample

this

method

is

insensitive

to

differences

in

extraction

efficiency

and

eliminates

the

need

for

control

genes

or

quantification

of

total

RNA

recovery

Figure

Models

of

regulatory

divergence

A

hypothetical

regulatory

network

is

shown

with

filled

circles

representing

regulatory

genes

open

circles

representing

structural

genes

-LRB-

all

with

altered

expression

-RRB-

and

lines

representing

regulatory

interactions

Crosses

indicate

genes

harbouring

a

regulatory

change

a

b

Two

extreme

models

of

regulatory

divergence

in

which

either

trans-regulatory

differences

-LRB-

a

-RRB-

or

cis-regulatory

differences

-LRB-

b

-RRB-

are

solely

responsible

for

expression

differences

c

A

distribution

of

cis

and

transregulatory

changes

consistent

with

our

data

NATURE

VOL

JULY

Statistical

analysis

After

normalization

replicate

pools

were

compared

for

each

gene

by

using

analysis

of

variance

Significant

heterogeneity

between

pools

was

observed

for

of

parental

pools

but

for

only

of

hybrid

pools

-LRB-

Supplementary

Fig.

-RRB-

This

variation

can

be

caused

either

by

residual

genetic

variation

within

inbred

strains

or

more

probably

by

environmental

variation

introduced

during

fly

rearing

However

because

no

single

pool

Nature

Publishing

Group

letters

to

nature

was

a

consistent

outlier

for

all

genes

data

from

all

pools

were

included

in

our

analysis

Differences

between

parental

pools

increase

our

confidence

intervals

for

the

interspecific

expression

difference

and

can

reduce

our

power

for

detecting

trans-regulatory

differences

To

deal

with

this

issue

interspecific

expression

differences

for

genes

with

unusually

large

variance

between

the

four

original

pools

were

measured

in

an

additional

three

parental

pools

The

normalization

procedure

used

to

correct

cDNA

measurements

for

experimental

bias

-LRB-

Supplementary

Information

-RRB-

prohibits

a

standard

nested

analysis

of

variance

but

a

t-test

provided

a

simple

and

robust

test

of

our

hypotheses

Two-tailed

t-tests

were

used

to

identify

cis-regulatory

divergence

-LRB-

-RRB-

interspecific

expression

differences

-LRB-

Mel/Sim

-RRB-

and

parent-of-origin

effects

-LRB-

in

reciprocal

crosses

-RRB-

To

identify

trans-regulatory

divergence

two-sided

t-tests

-LRB-

with

the

Cochran

correction

for

unequal

variances

-RRB-

and

nonparametric

Mann

Whitney

U-tests

were

used

to

compare

relative

expression

between

hybrid

and

parental

pools

The

decision

to

accept

or

reject

the

hypothesis

-LRB-

Mel/Sim

-RRB-

was

the

same

for

both

tests

for

all

except

three

genes

and

t-test

significance

was

ultimately

used

to

infer

trans-regulatory

divergence

All

statistical

analyses

were

preformed

with

SAS

software

v.

-LRB-

SAS

Institute

Cary

North

Carolina

-RRB-

and

are

shown

in

Supplementary

Table

Received

February

accepted

June

doi

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E.

H.

Genomic

Regulatory

Systems

Development

and

Evolution

-LRB-

Academic

San

Diego

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B.

Grenier

J.

K.

Weatherbee

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D.

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Diversity

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Animal

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Science

Oxford

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G.

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Altshuler

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H.

Yuan

W.

Velculescu

V.

E.

Vogelstein

B.

Kinzler

K.

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Allelic

variation

in

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Science

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-RRB-

Powell

J.

R.

Progress

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Prospects

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Evolutionary

Biology

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-LRB-

Oxford

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Pastinen

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et

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A

survey

of

genetic

and

epigenetic

variation

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human

gene

expression

Physiol

Genomics

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-RRB-

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A.

et

al.

Single-nucleotide

polymorphism

analysis

by

pyrosequencing

Anal

Biochem

-LRB-

-RRB-

Neve

B.

et

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SNP

allele

frequency

determination

in

genomic

DNA

pools

by

pyrosequencing

Biotechniques

-LRB-

-RRB-

Rifkin

S.

A.

Kim

J.

White

K.

P.

Evolution

of

gene

expression

in

the

Drosophila

melanogaster

subgroup

Nature

Genet

-LRB-

-RRB-

Ranz

J.

M.

Castillo-Davis

C.

I.

Meiklejohn

C.

D.

Hartl

D.

L.

Sex-dependent

gene

expression

and

evolution

of

the

Drosophila

transcriptome

Science

-LRB-

-RRB-

Davidson

E.

H.

McClay

D.

R.

Hood

L.

Regulatory

gene

networks

and

the

properties

of

the

developmental

process

Proc

Natl

Acad

Sci

USA

Ashburner

M.

et

al.

Gene

ontology

tool

for

the

unification

of

biology

The

Gene

Ontology

Consortium

Nature

Genet

-LRB-

-RRB-

The

FlyBase

Consortium

The

FlyBase

database

of

the

Drosophila

genome

projects

and

community

literature

Nucleic

Acids

Res

-LRB-

-RRB-

Yvert

G.

et

al.

Trans-acting

regulatory

variation

in

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cerevisiae

and

the

role

of

transcription

factors

Nature

Genet

-LRB-

-RRB-

Brem

R.

B.

Yvert

G.

Clinton

R.

Kruglyak

L.

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dissection

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in

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yeast

Science

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-RRB-

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E.

et

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man

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-LRB-

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J.

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Clark

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respiration

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Genetics

-LRB-

-RRB-

Davis

A.

W.

et

al.

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of

hybrid

sterility

in

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between

D.

melanogaster

and

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simulans

Nature

-LRB-

-RRB-

Sawamura

K.

Yamamoto

M.

T.

Watanabe

T.

K.

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lethal

systems

in

the

Drosophila

melanogaster

species

complex

II

The

Zygotic

hybrid

rescue

-LRB-

Zhr

-RRB-

gene

of

D.

melanogaster

Genetics

-LRB-

-RRB-

Hollocher

H.

Agopian

K.

Waterbury

J.

O'Neill

R.

W.

Davis

A.

W.

Characterization

of

defects

in

adult

germline

development

and

oogenesis

of

sterile

and

rescued

female

hybrids

in

crosses

between

Drosophila

simulans

and

Drosophila

melanogaster

J.

Exp

Zool

-LRB-

-RRB-

Barbash

D.

A.

Roote

J.

Ashburner

M

The

Drosophila

melanogaster

hybrid

male

rescue

gene

causes

inviability

in

male

and

female

species

hybrids

Genetics

-LRB-

-RRB-

Otto

P.

Kephart

D.

Bitner

R.

Huber

S.

Volkerding

K.

Separate

isolation

of

genomic

DNA

and

total

RNA

from

single

samples

using

the

SV

Total

RNA

Isolation

System

Promega

Notes

-LRB-

-RRB-

Evidence

for

dynamically

organized

modularity

in

the

yeast

protein

protein

interaction

network

Jing-Dong

J.

Nicolas

Tong

Debra

S.

Gabriel

F.

Lan

V.

Denis

Albertha

J.

M.

Michael

E.

Frederick

P.

Marc

Center

for

Cancer

Systems

Biology

and

Department

of

Cancer

Biology

Dana-Farber

Cancer

Institute

and

Department

of

Genetics

Harvard

Medical

School

Boston

Massachusetts

USA

Department

of

Biological

Chemistry

and

Molecular

Pharmacology

Harvard

Medical

School

Boston

Massachusetts

USA

Present

address

Program

in

Gene

Function

and

Expression

University

of

Massachusetts

Medical

School

Worcester

Massachusetts

USA

Supplementary

Information

accompanies

the

paper

on

Acknowledgements

We

thank

A.

Kumari

and

S.

Madhavarapu

for

experimental

assistance

K.

Montooth

for

statistical

advice

and

K.

Montooth

B.

Payseur

A.

Fiumera

T.

Schlenke

and

E.

Hill

for

comments

on

the

manuscript

Funding

for

this

project

was

provided

by

NIH

grants

to

A.G.C.

P.J.W.

is

a

Damon

Runyon

Fellow

supported

by

the

Damon

Runyon

Cancer

Research

Foundation

and

B.K.H.

was

funded

by

a

Howard

Hughes

Undergraduate

Research

award

Competing

interests

statement

The

authors

declare

that

they

have

no

competing

financial

interests

Correspondence

and

requests

for

materials

should

be

addressed

to

P.W.

-LRB-

-RRB-

In

apparently

scale-free

protein

protein

interaction

networks

or

interactome

most

proteins

interact

with

few

partners

whereas

a

small

but

significant

proportion

of

proteins

the

hubs

interact

with

many

partners

Both

biological

and

nonbiological

scale-free

networks

are

particularly

resistant

to

random

node

removal

but

are

extremely

sensitive

to

the

targeted

removal

of

A

link

between

the

potential

scale-free

topology

of

interactome

networks

and

genetic

seems

to

exist

because

knockouts

of

yeast

encoding

hubs

are

approximately

threefold

more

likely

to

confer

lethality

than

those

of

Here

we

investigate

how

hubs

might

contribute

to

robustness

and

other

cellular

properties

for

protein

protein

interactions

dynamically

regulated

both

in

time

and

in

space

We

uncovered

two

types

of

hub

party

hubs

which

interact

with

most

of

their

partners

simultaneously

and

date

hubs

which

bind

their

different

partners

at

different

times

or

locations

Both

in

silico

studies

of

network

connectivity

and

genetic

interactions

described

in

vivo

support

a

model

of

organized

modularity

in

which

date

hubs

organize

the

proteome

connecting

biological

processes

or

to

each

other

whereas

party

hubs

function

inside

modules

The

biological

role

of

topological

hubs

so

far

considered

in

static

representations

of

interactome

networks

without

information

on

the

functional

states

of

these

networks

that

is

dynamic

or

steady

might

vary

depending

on

the

timing

and

location

of

the

interactions

they

mediate

-LRB-

Fig.

-RRB-

Because

accurate

temporal

parameters

are

not

yet

available

for

many

protein

protein

interactions

we

estimated

temporal

characteristics

of

hubs

and

their

partners

by

using

compilations

of

yeast

messenger

RNA

expression

profiling

Hubs

connected

by

false-positive

interactions

would

be

uncorrelated

in

mRNA

expression

with

their

interaction

and

would

resemble

date

hubs

To

minimize

false

positives

we

first

generated

a

high-quality

yeast

interaction

data

set

by

intersecting

data

generated

by

several

different

interaction

detection

methods

-LRB-

see

Methods

-RRB-

The

resulting

filtered

yeast

interactome

-LRB-

FYI

-RRB-

data

set

contains

high-confidence

interactions

each

observed

by

at

least

two

different

methods

-LRB-

Supplementary

Fig.

-RRB-

FYI

is

a

high-quality

network

enriched

for

genuine

positives

-LRB-

Supplementary

Information

and

Supplementary

Fig.

-RRB-

The

FYI

network

contains

proteins

with

an

average

degree

of

interactions

per

protein

and

a

large

connected

component

of

proteins

Its

degree

distribution

follows

the

power

law

that

characterizes

scale-free

networks

-LRB-

Supplementary

Fig.

-RRB-

FYI

hubs

were

characterized

with

an

expression-profiling

compendium

of

data

points

for

most

yeast

genes

across

five

different

experimental

conditions

-LRB-

referred

to

below

as

the

yeast

expression

compendium

-RRB-

For

each

hub

we

calculated

the

average

of

Pearson

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O B O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O B O O O O O O O O O O O B O O O O O B O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

drosophila hybrids evolution regulatory expression transcription