The role of PET/CT in Cogan 's syndrome We report on the case of a 60-year-old woman with complaints of fatigue , coughing , anorexia , atypical chest pain , recurrent fever , and also ear pain and hearing loss . A test for anti-neutrophil cytoplasmic antibody -LRB- ANCA -RRB- was myeloperoxidase positive with p-ANCA specificity . Laboratory acute phase parameters were increased . A 2-deoxy-2 - -LSB- 18F -RSB- fluoro-d-glucose positron emission tomography/computed tomography investigation showed pathological uptake in the aorta ascendens , with no other involvement of the large vessels . After therapy with methylprednisolon intravenously and later prednisolon orally with methothrexate , her general condition and hearing loss improved both subjectively and objectively . `` Atypical '' Cogan 's syndrome was diagnosed on the basis of sensorineural deafness with improvement on steroids and large-vessel vasculitis of the aortic arch . Introduction The association of nonsyphilitic interstitial keratitis and audiovestibular involvement was first reported in 1934 by Mogan and Baumgartner . Their description was extended several years later by Cogan -LSB- 1 -RSB- . In `` typical '' Cogan 's syndrome , the presence of interstitial keratitis is necessary , the term `` atypical '' Cogan 's syndrome is used when other types of inflammatory eye disease , including conjunctivitis , uveitis , scleritis , and choroiditis are associated with the vestibuloauditory abnormalities -LSB- 2 -RSB- . In many cases , the symptomatology is not only restricted to the eyes and the ears but also other organs , thus resembling systemic vasculitis in one third of the patients . The most common symptoms are cardiovascular , musculoskeletal , neurological , gastrointestinal , and mucocutaneous -LSB- 3 -RSB- . Positron emission tomography -LRB- PET -RRB- with 2-deoxy-2 - -LSB- 18F -RSB- fluoro-d-glucose -LRB- FDG -RRB- is becoming increasingly important in diagnosis , staging , and therapy monitoring in clinical oncology and has recently been used in the diagnosis of infectious diseases with elevated intracellular glucose metabolism . Activated inflammatory cells have been shown to overexpress glucose transporters and to accumulate increased amounts of glucose and structurally related substances such as F18-FDG -LSB- 4 , 5 -RSB- . Therefore FDG-PET is also introduced as a diagnostic means to assess involvement in large vessel vasculitis -LSB- 6 -RSB- . In this report , we report the use of FDG-PET/computed tomography -LRB- CT -RRB- scanning in the diagnosis of Cogan 's syndrome . Case report A 60-year-old Caucasian woman was admitted to the hospital with a 4-month history of excessive fatigue , coughing , anorexia and weight loss , night sweats , and atypical chest pain . She also experienced short periods of fever . She experienced headaches and ear pain and hearing loss for over the last month , mainly on the left side , and felt sometimes dizzy . No blurred vision complaints or eye problems were noted . She was not known with any allergies . For her hypothyroidism -LRB- multinodular goiter -RRB- , she used Thyrax -LRB- l-thyroxine -RRB- 150 mcg once a day . She did not smoke and consumed alcohol only moderately . The family history revealed a daughter with systemic lupus erythematosus . Physical examination revealed a pulse of 104 , and bloodpressure was 125/85 mmHg and the temperature 37.1 °C . Heart sounds were normal , and the lungs were clear . The outer ears were normal . No lymphadenopathy was detected and no scalp tenderness or decreased pulsation at the temporal arteries was noted . Laboratory tests revealed an erythrocyte sedimentation rate -LRB- ESR -RRB- of 51 mm/h and C-reactive protein -LRB- CRP -RRB- of 53 mg/L . Test for rheumatoid factor was 42 kU/L -LRB- < 10 -RRB- , and tests for antinuclear facor and double-stranded DNA antibodies were negative . A test for anti-neutrophil cytoplasmic antibody -LRB- ANCA -RRB- appeared to be myeloperoxidase positive with p-ANCA specificity . Serum electrolytes and creatinine were normal . Her differential blood count , alkaline phosphatase , and transaminases were normal . The urine gave a negative test for protein , and the sediment contained no white cells , red cells , or casts in the urine . Initially arteritis temporalis was suspected , but Doppler ultrasonography of the temporal arteries showed no abnormalities . A chest X-ray revealed no interstitial or focal abnormalities . Subsequently , a PET/CT was performed , which showed pathological uptake in the wall of the aortic arch . More intens pathological uptake was seen at the beginning of the aorta descendens in the lateral wall , most likely a sign of perivascular inflammation . Maximum standard uptake value -LRB- SUV max -RRB- measured 11.9 . No other involvement of large vessels was noted -LRB- Fig. 1 -RRB- . A magnetic resonance imaging -LRB- MRI -RRB- scan of the cerebrum showed abnormalities suspicious for bilateral mastoiditis , possibly as a consequence of bilateral otitis media . Fig. 1 a Transverse fused PET/CT slice showing pathological uptake in the wall of the aortic arch and particularly in the lateral wall and perivascular space adjacent to the truncus pulmonalis -LRB- SUV max 11.9 -RRB- . b Follow-up PET/CT 3 weeks later , after treatment with methylprednisolon i.v. and prednisolon orally , showing clearly decreased uptake in the aortic arch -LRB- SUV max 4.3 -RRB- . c Second follow-up PET/CT 6 months later , while patient was in a stable condition with methotrexate and low-dose prednisone . Again , high pathological uptake in the aortic arch with higher intensity in the lateral wall and perivascular space adjacent to the truncus pulmonalis -LRB- SUV max 12.9 -RRB- After placing inner ear tubes , her hearing loss improved only little . Culture of the ear secretion was negative for pathogenic microorganisms . Audiograms confirmed sensorineural hearing loss , particularly in the left ear . Investigation of the eyes revealed no abnormalities , especially no keratitis . Therapy was started with three cycles of 1,000 mg methylprednisolon intravenously and later 60 mg prednisolon daily orally . Her general condition and hearing loss improved subjectively . Audiograms 6 weeks later showed also objective improvement of hearing . A control CT scan showed improvement of the abnormalites in both mastoid regions . As a consequence of the high doses of steroids , risedronate and calcium supplementation were started , and also methothrexate was added for the reason of its steroid-sparing effect . `` Atypical '' Cogan 's syndrome was diagnosed on the basis of sensorineural deafness with improvement on steroids and large-vessel vasculitis of the aortic arch . A follow-up PET/CT investigation 3 weeks later showed clearly decreased uptake in the aortic arch , especially a dramatic decrease at the location of the focal uptake in the lateral wall , compared to the first PET/CT . The SUV max was calculated 4.3 . ESR and CRP after 2 weeks of therapy were , respectively , 11 mm/h and < 2 mg/L . White cell count was still elevated -LRB- 16.9.109 L -RRB- . Six months later , she was in a stable condition with methotrexate and low-dose prednisone , and her ESR , CRP , and white cell count were now , respectively , 24 mm/h , 14 mg/L , and 13.7.109 L . A third PET/CT showed higher pathological activity , compared to the first PET/CT , in the wall of the aortic arch and in the perivascular space adjacent to the truncus pulmonalis -LRB- the SUV max was12 .9 compared to the SUV max of 11.9 of the first pretherapy PET/CT investigation -RRB- . Consequently , the dose of methothrexate and prednisone were both increased to 20 mg/day . Discussion The etiology of Cogan 's syndrome is unknown ; a minority of patients have rheumatoid factor , antinuclear bodies , and diminished complement levels . Histology of biopsies shows often vasculitis and perivascular inflammation . Giant cells may be present -LSB- 7 -RSB- . Specific involvement of large arteries has been reported in Cogan 's syndrome -LSB- 8 -RSB- . Aortitis in Cogan 's syndrome is indistinguishable from Takayasu 's arteritis . Cardiac involvement during the course of Cogan 's syndrome is , above all , aortic insufficiency . It is a severe complication that may require valve replacement , without which left ventricle involvement insufficiency develops , which can be fatal -LSB- 9 , 10 -RSB- . Standard diagnostic modalities such as biopsy , angiography , ultrasound , and MRI are commonly unable to demonstrate the full extent of vascular involvement in large-vessel vasculitis . PET investigations might play an important role here as large-vessel F18-FDG uptake is positively correlated with the level of acute phase reactant markers in patients with large vessel vasculitis . In a study of 18 patients with Takayasu 's arteritis , the F18-FDG-PET examination showed a sensitivity of 92 % and a specificity of 100 % -LSB- 11 -RSB- . This is , to the best of our knowledge , the first report of Cogan 's syndrome diagnosed by the use of FDG-PET/CT scanning . This case report is also supportive in the hypothesis that with PET , the inflammatory activity of large-vessel vasculitis is more accurately assessed compared to laboratory acute phase parameters -LSB- 12 -RSB- . The CT component of PET/CT is useful in the precise anatomical localization of the PET abnormalities and may provide information about changes in the wall structure or luminal flow -LSB- 13 -RSB- . The conclusion seems justified that F18-FDG-PET/CT is helpful in risk assessment of large-vessel vasculitis , as it provides intrinsically fused morphologic and functional data in a single examination .

O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

cogan’s syndrome
f18-fdg pet/ct
large-vessel vasculitis


Regulation

of

the

Xenopus

promoter

by

co-operating

VegT

and

sites

The

gene

encoding

the

Sox

F-group

transcription

factor

is

specifically

expressed

throughout

the

entire

region

of

the

Xenopus

blastula

fated

to

become

endoderm

and

is

important

in

controlling

endodermal

development

is

a

direct

target

of

the

maternal

endodermal

determinant

VegT

and

of

itself

We

have

analysed

the

promoter

of

the

Xenopus

laevis

gene

by

transgenesis

and

have

identified

two

important

control

elements

which

reside

about

kb

upstream

at

the

start

of

transcription

These

elements

individually

drive

transgenic

endodermal

expression

in

the

blastula

and

gastrula

One

contains

functional

cooperating

VegT

and

Sox-binding

consensus

sites

The

Sox

sites

in

this

region

are

occupied

in

vivo

The

other

responds

to

TGF-β

signals

like

Activin

or

Nodals

that

act

through

We

propose

that

these

two

regions

co-operate

in

regulating

the

early

endodermal

expression

of

the

gene

Introduction

The

endoderm

of

Xenopus

embryo

arises

in

two

successive

phases

involving

firstly

cell-autonomous

gene

action

followed

by

dependence

on

cell

signalling

The

cell-autonomous

phase

is

directly

initiated

by

the

maternal

T-box

transcription

factor

VegT

but

then

some

key

genes

associated

with

endodermal

differentiation

become

dependent

on

signalling

by

the

group

of

TGF-β

family

members

that

signal

through

These

include

the

Nodal-related

proteins

or

Xnrs

Derrière

and

Activin

In

this

second

phase

cells

are

sensitised

to

TGF-β

signalling

by

the

maternal

VegT

that

they

inherit

-LRB-

Clements

et

al.

Clements

and

Woodland

Engleka

et

al.

Hudson

et

al.

Yasuo

and

Lemaire

-RRB-

Since

the

essential

TGF-β

signalling

molecules

are

themselves

induced

by

VegT

and

VegT

is

indispensable

for

endoderm

development

-LRB-

Xanthos

et

al.

-RRB-

the

overall

effect

is

that

only

a

critical

mass

of

VegT-containing

cells

can

generate

sufficient

signalling

to

sustain

expression

of

these

key

endodermal

genes

Scattered

VegT

containing

cells

will

fail

to

become

endoderm

and

conform

to

their

surroundings

In

contrast

some

other

genes

-LRB-

e.g.

-RRB-

which

are

directly

induced

by

VegT

do

not

become

signal-dependent

Finally

in

the

gastrula

endodermal

gene

expression

becomes

independent

of

cell

signalling

-LRB-

Yasuo

and

Lemaire

-RRB-

This

interpretation

of

endoderm

initiation

establishment

and

maintenance

is

heavily

based

on

studying

the

expression

of

the

VegT

target

an

HMG-box

transcription

factor

although

Mix

and

Mixer

behave

in

a

similar

fashion

There

are

three

genes

in

Xenopus

laevis

α2

and

β

but

since

no

differences

in

their

activity

have

so

far

been

detected

for

most

purposes

we

refer

to

them

collectively

as

The

transcription

of

these

Sox

F

group

genes

is

activated

prior

to

the

mid-blastula

transition

-LRB-

MBT

-RRB-

when

very

low

levels

are

found

ubiquitously

but

transcription

in

the

vegetal

pole

is

enormously

upregulated

at

MBT

precisely

marking

out

the

territory

of

the

future

endoderm

through

late

blastula

gastrula

and

neurula

stages

-LRB-

Hudson

et

al.

Zorn

et

al.

-RRB-

The

genes

have

a

key

role

in

endoderm

formation

Their

ectopic

expression

induces

endodermal

gene

expression

as

well

as

changing

the

fate

of

cells

Interfering

with

their

expression

with

a

dominant

negative

Engrailed

fusion

construct

has

the

converse

effect

inhibiting

endodermal

gene

expression

and

shifting

cells

out

of

an

endodermal

fate

in

intact

embryos

-LRB-

Clements

and

Woodland

Hudson

et

al.

-RRB-

The

use

of

antisense

morpholino

oligos

shows

that

the

individual

genes

have

non-redundant

late

roles

in

the

developing

mid

and

hindgut

but

that

together

they

are

needed

for

the

completion

of

gastrulation

-LRB-

Clements

et

al.

-RRB-

This

correlates

well

with

the

essential

role

of

the

single-murine

gene

for

early

development

of

endoderm

fated

to

become

mid

and

hindgut

although

in

the

absence

of

there

is

also

later

loss

of

foregut

cells

-LRB-

Kanai-Azuma

et

al.

-RRB-

In

zebrafish

two

related

Sox

genes

Casanova

and

are

important

in

forming

the

endoderm

the

former

is

more

upstream

and

its

mutants

indicate

that

it

has

a

vital

role

in

endoderm

development

-LRB-

Aoki

et

al.

-RRB-

While

mutations

in

the

zebrafish

gene

have

not

been

described

it

is

likely

that

in

the

zebrafish

endodermal

gene

network

the

combined

action

of

Casanova

and

are

roughly

equivalent

to

the

overall

action

of

in

Xenopus

particularly

allowing

for

the

fact

that

Casanova

expression

in

the

yolk

syncytial

layer

is

Nodal-independent

allowing

a

parallel

with

the

Xenopus

cell

autonomous

phase

to

be

drawn

-LRB-

Kikuchi

et

al.

Woodland

and

Clements

-RRB-

In

Xenopus

ablation

of

expression

with

morpholinos

halts

gastrulation

at

an

early

stage

However

the

immediate

effects

on

gene

expression

were

initially

reported

to

be

modest

and

were

restricted

to

the

direct

targets

Endodermin

and

-LRB-

Clements

et

al.

-RRB-

However

much

wider

effects

have

now

been

observed

using

microarrays

-LRB-

Sinner

et

al.

-RRB-

Since

is

a

crucial

gene

in

the

early

endoderm

and

its

expression

defines

the

endodermal

territory

we

have

analysed

the

regulatory

elements

in

the

promoter

approximately

kb

upstream

of

transcriptional

initiation

We

have

identified

two

small

elements

which

can

confer

endodermal

expression

on

a

reporter

gene

in

the

early

embryo

We

have

analysed

one

of

these

in

detail

and

show

that

its

activity

depends

on

co-operating

VegT

and

sites

whereas

the

other

regulatory

region

responds

to

Activin

Materials

and

methods

Biological

materials

Eggs

of

X.

laevis

were

obtained

fertilised

dejellied

and

cultured

by

standard

methods

as

described

previously

-LRB-

Wilson

et

al.

-RRB-

Oocytes

complete

with

their

follicle

were

manually

removed

using

watchmakers

forceps

in

modified

Barths

saline

-LRB-

MBS

mM

NaCl

mM

KCl

mM

mM

Ca

-LRB-

-RRB-

mM

mM

mM

Tris

HCl

pH

-RRB-

Transgenic

methods

These

followed

the

procedure

of

Kroll

and

Amaya

-LRB-

-RRB-

except

that

a

reduced

amount

of

egg

extract

was

used

in

the

incubation

of

stored

frozen

sperm

nuclei

with

DNA

-LRB-

μl

in

a

μl

reaction

-RRB-

The

reaction

typically

contained

ng

of

linearised

plasmid

DNA

and

μl

of

a

dilution

of

restriction

enzyme

-LRB-

units/ml

-RRB-

Injections

of

dejellied

unfertilised

eggs

were

performed

in

Ficoll

Marc

's

modified

Ringer

-LRB-

MMR

-RRB-

or

MBS

in

polyheme-coated

plastic

dishes

Correctly

cleaving

eggs

were

sorted

at

the

to

stage

and

incubated

in

Ficoll

modified

Barth

's

medium

GFP

fluorescence

was

monitored

using

a

Leica

MZFLIII

dissecting

microscope

Transient

transgenesis

in

embryos

and

oocytes

For

transient

expression

in

embryos

DNA

constructs

were

linearised

and

purified

with

the

Qiagen

gel

extraction

kit

pg

DNA

together

with

pg

control

Renilla

luciferase

reporter

in

nl

water

were

injected

bilaterally

with

or

without

mRNA

into

the

animal

or

vegetal

poles

of

embryos

cultured

in

Ficoll

MBS

Embryos

were

analysed

at

gastrula

or

neurula

stages

using

the

Promega

Dual

Luciferase

Reporter

Assay

System

Triplicate

batches

of

embryos

were

homogenised

in

μl

passive

lysis

buffer

and

incubated

on

ice

for

min

Samples

were

centrifuged

at

rpm

for

min

at

°C

and

supernatants

removed

They

were

equilibrated

to

room

temperature

for

min

and

μl

assayed

for

bioluminescence

after

addition

of

μl

luciferin

stock

using

the

Luminoskan

RS

apparatus

-LRB-

Labsystems

-RRB-

Normalisation

of

the

experimental

reporter

was

achieved

by

quenching

of

the

firefly

luciferase

reaction

and

measurement

of

Renilla

luciferase

luminescence

Oocyte

nuclei

were

microinjected

with

nl

water

containing

pg

circular

firefly

test

plasmid

plus

one

third

this

amount

of

control

Renilla

luciferase

plasmid

with

or

without

transcription

factor

mRNA

After

culturing

overnight

in

MBS

they

were

processed

for

luciferase

activity

as

detailed

above

Cloning

and

characterisation

of

the

gene

The

gene

was

isolated

by

screening

a

X.

laevis

gilli

PAC

library

-LRB-

RZPD

-RRB-

with

an

cDNA

probe

One

clone

-LRB-

-RRB-

reacted

strongly

with

this

probe

A

positive

Not

fragment

was

subcloned

into

Bluescript

and

this

sequence

encompassed

all

kb

of

upstream

sequence

present

in

the

PAC

the

transcribed

region

itself

and

kb

downstream

of

the

UTR

Transgenic

constructs

and

mutagenesis

Mutant

promoter

constructs

were

created

by

hybrid

overlap

extension

PCR

using

a

series

of

external

and

overlapping

internal

primers

Primer

sequences

were

as

follows

ext

up

CAACACTCACATTC

T-box

ext

down

CTTGAGAATGGGACTGTGTTAACAAACAATGATGATCAGAACTCTGG

Sox

A

int

up

CTTGGGAACTAGTTGTGGATC

Sox

A

int

down

GATCCACAACTAGTTCCCAAG

Sox

B

int

down

CTTGAGAATGGGACTGTGTTAACAACCATGGATGGTGTGAACTCTGG

Sox

B

T-box

ext

down

CTTGAGAATGGGACTGTTTTAACAACCATGGATGATCAGAACTCTGG

Underlined

text

denotes

mutated

sequence

External

primers

had

and

extension

sequences

Amplification

was

performed

over

cycles

with

°C

annealing

temperature

Products

were

Qiagen

column

purified

cut

with

restriction

enzyme

and

cloned

into

-LRB-

transient

assay

-RRB-

or

-LRB-

transgenic

assay

-RRB-

Electrophoretic

band-shifts

-LRB-

EMSAs

-RRB-

EMSAs

were

performed

using

VegT

protein

synthesised

in

vitro

in

the

rabbit

reticulocyte

system

pmol

single-stranded

DNA

oligo

was

end-labelled

with

μCi

γ-ATP

using

polynucleotide

kinase

Forward

and

unlabelled

reverse

strands

were

annealed

by

heating

to

°C

for

min

followed

by

slow

cooling

overnight

DNA

was

recovered

by

ethanol

precipitation

and

resuspended

in

TE

buffer

μl

binding

reactions

contained

μl

in

vitro

translated

protein

μg

-LSB-

poly

dI.dC

-RSB-

-LSB-

poly

dI.dC

-RSB-

μl

MDB

buffer

-LRB-

mM

HEPES

pH

mM

KCl

mM

mM

EDTA

glycerol

w/v

mM

DDT

-RRB-

The

probe

was

added

following

incubation

at

°C

followed

by

a

further

incubation

Control

binding

reactions

included

a

excess

of

unlabelled

specific

competitor

probe

Samples

were

analysed

on

a

polyacrylamide

gel

at

°C

-LRB-

V

h

-RRB-

Gels

were

dried

and

autoradiographed

or

analysed

with

a

phosphoimager

Probe

sequences

used

were

T-box

mutant

T-box

consensus

Underlined

text

denotes

T-box

half

site

and

variations

thereof

Chromatin

immunoprecipitation

-LRB-

ChIP

-RRB-

gastrula

stage

embryos

were

fixed

in

formaldehyde

for

min

at

room

temperature

and

fixation

reversed

by

addition

of

mM

glycine

for

min

Embryos

were

washed

in

MMR

-LRB-

mM

NaCl

mM

KCl

mM

mM

mM

HEPES

pH

-RRB-

and

homogenised

in

μl

low

salt

extraction

buffer

-LRB-

mM

Tris

Cl

pH

mM

KCl

mM

EDTA

glycerol

mM

DTT

protease

inhibitors

-RRB-

Shearing

was

performed

by

sonication

at

full

power

for

s

with

breaks

on

ice

Shearing

efficiency

was

assessed

by

agarose

gel

electrophoresis

following

reversal

of

cross

links

by

the

addition

of

mM

NaCl

and

incubation

at

°C

for

h

followed

by

proteinase

K

treatment

phenol

extraction

and

ethanol

precipitation

This

preparation

also

yielded

input

DNA

for

quantification

of

PCR

reactions

μl

sheared

chromatin

preparation

was

diluted

with

μl

IP

buffer

-LRB-

mM

Tris

Cl

pH

mM

NaCl

mM

EDTA

mM

DTT

protease

inhibitors

-RRB-

and

pre-cleared

with

μl

protein

A

agarose

-LRB-

Sigma

-RRB-

for

hrs

at

°C

Supernatant

was

incubated

with

μl

serum

for

hrs

at

°C

A

no

antibody

control

was

also

included

μl

slurry

protein

A

agarose

pre-saturated

with

mg/ml

BSA

and

mg/ml

salmon

sperm

DNA

in

IP

buffer

was

added

and

reactions

incubated

overnight

at

°C

Beads

were

washed

successively

in

IP

buffer

plus

sodium

deoxycholate

IP

buffer

with

mM

NaCl

IP

buffer

with

mM

LiCl

and

TE

-LRB-

mM

Tris

pH

mM

NaCl

-RRB-

Following

a

pulse

spin

μl

elution

buffer

-LRB-

SDS

M

-RRB-

was

added

to

the

beads

and

elution

repeated

until

a

μl

volume

was

obtained

Cross-links

were

reversed

and

DNA

recovered

as

described

above

PCR

was

performed

as

described

previously

cycles

of

amplification

were

used

Primer

sequences

are

as

detailed

below

GCCAATAGACACCTTTCTAG

GAGAATGGGACTGTGTTAAC

ORF

GGACGAGTGCCAGATGATG

ORF

CTGGCAAGTACATGTGTCC

Xom

Promoter

TGTTGGCTGAGTAGGAATGAGAGG

Xom

Promoter

AGGCAGAGATCAGTACCACCT

The

Xom

primers

are

from

Messenger

et

al.

-LRB-

-RRB-

Results

Structure

of

the

gene

The

gene

was

isolated

from

a

X.

laevis

gilli

PAC

library

-LRB-

RZPD

-RRB-

using

an

cDNA

probe

A

Not

fragment

that

encompassed

all

kb

of

upstream

sequence

present

in

the

PAC

and

kb

downstream

of

the

UTR

was

subcloned

into

Bluescript

The

transcribed

sequence

contains

a

single

intron

of

bp

starting

in

codon

The

upstream

region

was

sequenced

up

to

kb

although

sequence

from

approximately

to

kb

was

highly

repetitive

and

proved

unsequenceable

The

transcriptional

start

site

was

determined

by

primer

extension

-LRB-

data

not

shown

-RRB-

In

parallel

an

phage

λ

clone

with

kb

of

upstream

sequence

was

isolated

and

all

of

its

regions

sequenced

Since

the

alpha

and

beta

genes

have

identical

early

expression

patterns

a

comparison

was

made

of

the

proximal

promoter

regions

-LRB-

Supplementary

Fig.

-RRB-

There

are

many

conserved

features

including

motifs

for

binding

several

transcription

factors

known

to

be

relevant

to

endoderm

development

-LRB-

Homeodomain

SMAD

T-box

and

Sox

proteins

-RRB-

as

well

as

a

GA-rich

region

at

about

bp

No

other

or

clones

were

identified

in

any

library

Transgenic

analysis

of

the

promoter

of

the

gene

Initially

the

entire

kb

upstream

of

the

gene

was

fused

to

a

GFP

expression

cassette

to

give

A

second

construct

was

made

which

also

contained

kb

downstream

of

the

transcribed

sequence

This

was

placed

downstream

of

the

GFP

cassette

-LRB-

-RRB-

-LRB-

Fig.

top

panel

-RRB-

When

the

method

of

Kroll

and

Amaya

-LRB-

-RRB-

was

used

to

make

transgenics

containing

these

sequences

GFP

expression

was

observed

throughout

the

pre-endoderm

of

the

gastrula

including

the

superficial

region

surrounding

the

blastopore

lip

which

is

known

as

the

involuting

or

extra-blastoporal

pre-endoderm

-LRB-

Figs.

B

-RRB-

Expression

was

screened

by

GFP

fluorescence

which

under-reports

in

the

vegetal

region

-LRB-

Ahmed

et

al.

-RRB-

and

it

was

confirmed

by

in

situ

hybridisation

to

GFP

mRNA

-LRB-

C

-RRB-

which

also

under-reports

in

vegetal

tissues

but

to

a

much

lesser

degree

if

suitable

protocols

are

used

-LRB-

Sasai

et

al.

-RRB-

Bearing

this

in

mind

Fig.

shows

that

the

pattern

at

early

and

late

gastrula

was

similar

to

the

in

situ

hybridisation

pattern

of

expression

of

the

endogenous

gene

-LRB-

E

F

-RRB-

Comparison

of

and

transgenics

indicated

that

they

were

similar

in

the

gastrula

-LRB-

H

-RRB-

but

that

the

region

of

reduced

expression

outside

the

endoderm

at

later

stages

This

sequence

has

not

been

investigated

further

The

bp

minimal

promoter

was

negative

in

terms

of

endodermal

expression

-LRB-

I

-RRB-

but

was

expressed

elsewhere

Typical

numerical

results

for

the

transgenics

are

shown

in

Supplementary

Tables

and

Constructs

either

consistently

gave

endodermal

expression

in

the

gastrula

or

consistently

failed

to

do

so

There

was

later

expression

of

GFP

in

the

pharynx

and

hindgut

of

the

tailbud

tadpole

-LRB-

Supplementary

Fig.

-RRB-

This

corresponds

to

regions

where

the

endogenous

gene

is

expressed

as

judged

by

RT

PCR

-LRB-

Clements

et

al.

-RRB-

but

there

was

no

expression

in

the

region

of

the

developing

gall

bladder

where

the

gene

is

strongly

expressed

at

this

stage

-LRB-

Hudson

et

al.

Zorn

and

Mason

-RRB-

Later

there

was

robust

expression

in

the

foregut

and

proctodeum

as

well

as

in

the

pancreas

-LRB-

Supplementary

Fig.

-RRB-

Zorn

and

Mason

-LRB-

-RRB-

did

not

report

expression

in

the

pancreas

at

these

stages

so

this

may

represent

either

incorrect

regulation

or

persistent

stable

GFP

expression

There

was

also

expression

in

lateral

line

organs

and

in

the

brain

-LRB-

fore/mid

brain

boundary

cranial

nerves

-RRB-

It

is

not

known

if

is

expressed

in

all

these

regions

However

there

was

GFP

expression

in

the

lens

and

RT

PCR

shows

that

is

expressed

there

as

are

other

Sox

genes

-LRB-

data

not

shown

-RRB-

-LRB-

Schlosser

and

Ahrens

Zygar

et

al.

-RRB-

There

was

also

expression

in

the

olfactory

organs

which

has

been

shown

for

the

endogenous

genes

by

in

situ

hybridisation

-LRB-

Zorn

personal

communication

-RRB-

as

well

as

in

skeletal

elements

of

the

developing

limbs

We

have

focused

on

blastula

to

early

gastrula

stage

expression

of

the

promoter

because

this

is

the

time

that

the

endodermal

domain

marked

by

expression

is

mapped

out

To

define

the

regions

responsible

we

first

showed

that

there

were

no

sequences

promoting

endodermal

expression

in

the

single

intron

or

the

kb

downstream

of

the

cDNA

sequence

-LRB-

not

shown

-RRB-

We

then

showed

that

although

the

bp

proximal

region

contains

sequences

conserved

in

the

promoter

including

T-box

sites

-LRB-

Supplementary

Fig.

-RRB-

it

drove

expression

everywhere

except

the

future

endoderm

It

is

thus

expressed

only

where

is

not

However

later

it

gave

expression

in

the

pharynx

duodenum

pancreas

stomach

and

proctodeum

as

illustrated

for

longer

promoters

in

Supplementary

Fig.

The

kb

upstream

of

also

did

not

drive

gastrula

endodermal

expression

-LRB-

not

shown

-RRB-

and

we

could

not

identify

any

clone

Thus

a

comparison

of

endodermal

elements

in

the

three

genes

could

not

be

made

While

it

is

possible

to

compare

sequences

with

those

in

Xenopus

tropicalis

any

interpretation

would

be

dubious

since

the

detailed

regulation

of

X.

tropicalis

has

not

been

studied

in

this

species

A

wider

screen

for

regulatory

elements

in

was

made

with

a

deletion

series

-LRB-

Fig.

-RRB-

Early

endodermal

expression

required

regions

about

kb

from

the

start

of

transcription

This

defined

a

kb

Endodermal

element

which

alone

drove

strong

GFP

expression

in

the

early

vegetal/endoderm

region

Internal

deletions

show

that

the

E-element

can

drive

strong

endodermal

expression

when

placed

on

the

first

kb

to

the

start

of

transcription

which

itself

only

gave

low

GFP

expression

in

non-endodermal

regions

of

gastrulae

and

later

stages

-LRB-

Fig.

-RRB-

Later

experiments

described

below

investigated

sub-fragments

of

this

region

fused

to

a

basal

promoter

from

a

Xenopus

cytoskeletal

actin

gene

that

is

expressed

in

embryonic

striated

muscle

-LRB-

Ahmed

et

al.

Latinkic

et

al.

-RRB-

showing

that

the

E-element

does

not

require

sequences

in

the

proximal

region

of

the

promoter

for

endoderm-specific

expression

Thus

in

the

context

of

the

sequences

studied

the

E-element

is

the

only

region

both

necessary

and

sufficient

to

direct

expression

in

the

progenitor

of

the

endoderm

and

in

the

early

gastrula

Dissection

of

the

E-element

The

E-element

was

divided

into

three

sub-fragments

-LRB-

A

C

-RRB-

which

were

coupled

to

the

basal

cytoskeletal

actin

promoter

and

tested

in

transgenics

-LRB-

Fig.

-RRB-

Only

B

and

C

gave

vegetal

expression

Each

was

further

divided

into

two

approximately

equal

fragments

For

B

only

drove

vegetal/endodermal

expression

but

neither

C

sub-fragment

did

However

a

sub-fragment

from

the

central

region

of

C

gave

vegetal

expression

albeit

with

relatively

high

animal

expression

-LRB-

Fig.

-RRB-

Since

the

and

fragments

were

made

with

an

overlapping

bp

region

to

avoid

disrupting

a

regulatory

sequence

there

clearly

are

sequences

within

and

which

must

co-operate

to

give

the

endodermal

expression

of

Thus

two

independent

control

regions

and

are

capable

of

driving

expression

in

the

presumptive

endoderm

of

the

gastrula

The

expression

of

-LRB-

Figs.

C

-RRB-

is

notably

similar

to

the

endogenous

genes

-LRB-

Figs.

F

-RRB-

The

GFP

expression

in

the

vegetal

regions

is

detectable

by

early

stage

the

onset

of

gastrulation

Given

that

GFP

requires

an

hour

or

more

to

mature

and

fluoresce

vegetal

expression

of

the

transgene

must

begin

in

the

blastula

during

the

initiation

and/or

establishment

phases

of

regulation

-LRB-

Clements

et

al.

Yasuo

and

Lemaire

-RRB-

As

discussed

earlier

GFP

under-reports

in

the

vegetal

pole

-LRB-

Ahmed

et

al.

-RRB-

so

any

detectable

vegetal

fluorescence

indicates

strong

actual

expression

Transgenic

analysis

of

the

T-box

and

Sox

sites

in

element

contains

several

consensus

binding

sites

for

known

endodermal

regulators

-LRB-

Fig.

Supplementary

Fig.

-RRB-

including

a

variant

T-box

binding

site

-LRB-

core

CACCA

rather

than

CACCT

-RRB-

and

two

canonical

Sox

sites

-LRB-

core

CATTG

-RRB-

To

test

if

the

variant

T-box

binds

VegT

we

performed

electrophoretic

gel

mobility

shift

assays

-LRB-

EMSA

-RRB-

comparing

the

binding

of

VegT

to

this

sequence

and

the

consensus

sequence

identified

in

the

Derrière

promoter

-LRB-

White

et

al.

-RRB-

Fig.

shows

that

the

binding

of

the

two

is

similar

As

a

control

no

binding

was

observed

to

a

sequence

containing

a

mutant

site

-LRB-

core

CACAT

-RRB-

The

T-box

and

the

two

Sox

sites

were

mutated

and

their

efficiency

in

transgenics

assessed

-LRB-

Fig.

-RRB-

The

numbers

of

transgenic

embryos

in

a

typical

experiment

are

shown

in

Supplementary

Table

The

double

Sox

site

mutant

gave

vegetal

pole

expression

which

was

somewhat

less

intense

than

that

produced

by

the

wild-type

There

was

also

raised

relative

expression

in

the

animal

pole

suggesting

that

these

sites

may

play

a

possible

repressive

role

in

the

non-endodermal

region

The

T-box

mutant

also

gave

vegetal

expression

although

again

it

appeared

to

be

at

level

lower

than

from

an

unmutated

element

although

this

is

difficult

to

quantify

When

the

T-box

site

was

mutated

in

conjunction

with

the

Sox

sites

-LRB-

Fig.

triple

mutant

-RRB-

expression

of

GFP

was

excluded

from

the

endodermal

domain

Transient

transgenic

analysis

of

and

Although

in

transgenics

the

constructs

either

gave

endodermal

expression

or

they

did

not

the

results

are

not

quantitative

and

the

variability

in

the

absolute

level

of

expression

from

embryo

to

embryo

makes

it

difficult

to

compare

constructs

objectively

Transient

transgenesis

using

the

quantifiable

reporter

luciferase

circumvents

this

problem

DNA

may

be

introduced

into

different

parts

of

the

embryo

or

injected

it

into

a

region

where

is

not

expressed

-LRB-

i.e.

the

animal

hemisphere

-RRB-

with

or

without

mRNAs

encoding

specific

regulatory

molecules

like

VegT

To

enable

this

promoter

elements

together

with

the

cytoskeletal

actin

basal

promoter

were

cloned

into

-LRB-

Promega

-RRB-

which

lacks

eukaryotic

promoter

and

enhancer

sequences

The

DNA

was

linearised

for

injection

because

sometimes

this

is

necessary

for

expression

-LRB-

Wilson

et

al.

-RRB-

First

constructs

containing

and

elements

were

injected

into

vegetal

and

animal

hemispheres

to

establish

whether

the

transient

expression

mirrors

the

natural

high

expression

of

in

the

vegetal

compared

to

the

animal

pole

Fig.

shows

that

is

much

more

active

in

the

vegetal

than

the

animal

hemisphere

in

agreement

with

the

transgenics

This

is

also

true

to

a

reduced

degree

of

the

combined

fragment

Surprisingly

showed

reduced

expression

in

the

vegetal

relative

to

the

animal

hemispheres

This

may

partly

be

because

these

results

are

expressed

as

a

ratio

of

animal

to

vegetal

expression

High

expression

in

the

animal

hemisphere

was

also

seen

in

GFP

transgenics

where

we

simply

scored

for

vegetal

expression

Presumably

inhibitory

elements

are

lacking

contains

T-box

and

Sox-binding

sites

that

were

essential

for

high

endodermal

expression

in

transgenics

We

tested

the

functionality

of

the

T-box

site

by

co-injecting

DNA

plus

VegT

mRNA

into

the

animal

pole

where

VegT

mRNA

is

normally

present

only

at

a

low

level

The

basal

promoter

did

not

respond

to

VegT

in

these

assays

-LRB-

not

shown

-RRB-

VegT

stimulates

activity

by

an

amount

comparable

to

that

produced

by

vegetal

compared

to

animal

hemisphere

injections

-LRB-

Fig.

-RRB-

In

contrast

is

inhibited

slightly

even

though

it

contains

a

consensus

T-box

site

core

sequence

-LRB-

CACCT

-RRB-

Flanking

nucleotides

of

this

site

are

divergent

-LRB-

AGACACCT

consensus

TCACACCT

-RRB-

and

White

et

al.

found

that

this

sequence

bound

VegT

only

weakly

in

EMSAs

-LRB-

White

et

al.

-RRB-

The

fragment

produced

an

intermediate

result

Thus

but

not

responds

positively

to

VegT

and

the

responses

of

and

to

VegT

mirror

their

activity

in

vegetal

versus

animal

poles

Since

VegT

directly

or

indirectly

induces

expression

of

many

transcription

factors

which

are

part

of

the

mesendodermal

gene

network

including

other

T-box

proteins

-LRB-

Eomes

and

Xbra

-RRB-

we

sought

to

confirm

that

VegT

directly

interacts

with

in

two

ways

It

is

of

course

known

that

the

endogenous

genes

are

direct

VegT

targets

from

experiments

with

inducible

VegT

fusions

-LRB-

Clements

et

al.

Clements

and

Woodland

Yasuo

and

Lemaire

-RRB-

Firstly

we

mutated

the

T-box

site

which

greatly

reduced

but

did

not

eliminate

the

stimulation

by

VegT

-LRB-

Fig.

lane

-RRB-

This

implicates

a

T-box

protein

in

the

regulation

Secondly

we

examined

expression

in

oocytes

in

response

to

co-injected

VegT

-LRB-

White

et

al.

-RRB-

The

amount

of

transcription

seen

when

DNA

is

injected

into

an

oocyte

nucleus

is

comparable

to

an

embryo

this

being

supported

by

stores

of

chromosomal

and

transcriptional

proteins

However

there

is

only

one

nucleus

in

an

oocyte

so

the

amount

of

downstream

nuclear

gene

expression

that

the

oocyte

can

support

over

the

time

course

of

our

experiments

is

negligible

even

over

an

time

course

-LRB-

White

et

al.

-RRB-

One

hour

of

transcription

in

the

late

blastula

would

be

equivalent

to

h

for

the

oocyte

equating

to

well

over

a

year

This

suggests

that

any

effects

of

VegT

on

the

co-injected

DNA

are

directly

dependent

on

VegT

itself

Fig.

-LRB-

last

three

lanes

-RRB-

shows

that

VegT

stimulates

transcription

in

oocytes

and

that

this

is

absent

for

the

T-box

mutant

The

stimulation

is

less

in

oocytes

than

in

embryos

presumably

because

cooperating

genes

are

not

significantly

induced

by

VegT

in

oocytes

For

example

endogenous

expression

is

dependent

both

on

VegT

itself

and

on

the

TGF-β

signalling

that

VegT

induces

-LRB-

Clements

et

al.

Clements

and

Woodland

Xanthos

et

al.

-RRB-

To

test

if

the

element

responds

to

TGF-βs

downstream

of

VegT

-LRB-

directly

or

indirectly

-RRB-

we

blocked

TGF-β

family

signalling

by

co-expressing

VegT

and

a

truncated

Activin

receptor

-LRB-

tXAR

-RRB-

in

animal

hemispheres

-LRB-

Hemmati-Brivanlou

and

Melton

-RRB-

Fig.

lane

shows

that

the

stimulation

of

by

VegT

is

reduced

but

not

eliminated

These

results

are

consistent

with

the

proposal

that

responds

to

VegT

directly

via

its

T-box

site

but

that

co-operating

molecules

downstream

of

VegT-induced

TGF-β

signalling

are

also

necessary

for

the

full

VegT

induction

in

an

embryo

Consistent

with

this

interpretation

in

oocytes

there

is

no

VegT

stimulation

of

the

element

with

a

mutated

T-box

because

VegT

can

not

induce

downstream

genes

such

as

TGF-β

signals

-LRB-

Fig.

last

lane

-RRB-

Transgenesis

also

showed

that

contained

functional

Sox-binding

sites

and

it

was

already

known

that

the

endogenous

genes

are

direct

targets

-LRB-

Sinner

et

al.

-RRB-

Fig.

shows

that

in

the

animal

hemisphere

responds

strongly

to

and

expression

in

the

oocyte

supports

the

view

that

this

is

a

direct

effect

and

β

induce

themselves

and

each

other

-LRB-

Sinner

et

al.

-RRB-

but

the

autoinduction

is

quite

slow

as

shown

in

Fig.

The

luciferase

inductions

fitted

this

because

there

was

no

significant

induction

by

at

stage

-LRB-

not

shown

-RRB-

but

it

was

clear

by

stage

-LRB-

h

later

at

°C

-RRB-

probably

normally

co-operates

with

other

factors

that

are

maternal

or

downstream

of

VegT

on

certain

promoters

However

this

only

applies

to

a

subset

of

promoters

whereas

other

genes

like

do

not

show

this

delay

presumably

because

any

co-operating

factors

are

already

present

in

the

animal

hemisphere

It

is

likely

that

when

alone

is

expressed

ectopically

it

slowly

induces

the

co-operating

factors

needed

for

auto-induction

eventually

establishing

a

partial

endodermal

gene

network

Normally

the

cooperating

molecules

are

maternal

or

are

induced

by

VegT

In

contrast

to

did

not

respond

to

as

expected

from

its

lack

of

consensus

Sox

sites

While

was

activated

by

neither

VegT

nor

it

contains

several

consensus

Smad-binding

sites

coupled

to

those

for

co-operating

-LRB-

-RRB-

suggesting

that

it

might

respond

to

TGF-βs

like

Activin

Fig.

shows

that

this

is

indeed

the

case

suggesting

that

it

might

mediate

the

TGF-β

response

of

In

oocytes

is

induced

by

constituently

active

indicating

that

the

induction

by

Activin

is

direct

-LRB-

Fig.

-RRB-

In

contrast

shows

very

little

response

to

Activin

in

embryos

This

is

surprising

since

many

gene

products

that

induce

are

downstream

of

Activin

including

the

themselves

While

in

this

experiment

the

luciferase

was

assayed

before

would

have

exerted

its

slow

effect

in

animal

caps

-LRB-

see

above

-RRB-

there

is

little

effect

even

at

later

time

points

-LRB-

not

shown

-RRB-

which

suggests

that

the

level

of

induced

by

Activin

is

too

low

to

produce

a

strong

induction

without

co-operating

VegT

We

show

below

that

VegT

and

co-operate

in

regulating

and

synergy

between

VegT

and

Activin

was

previously

noted

-LRB-

Clements

and

Woodland

-RRB-

Mutational

analysis

of

shows

that

one

Sox

site

is

most

important

and

it

co-operates

with

the

VegT

site

To

assess

the

roles

of

the

two

Sox

sites

in

the

response

of

a

series

of

single

double

and

triple

mutants

of

Sox

and

T-box

sites

was

assessed

by

injection

into

animal

hemispheres

Most

of

the

response

to

VegT

was

dependent

on

the

T-box

site

and

the

neighbouring

B

Sox

site

whereas

removal

of

the

more

distant

A

Sox

site

had

much

less

effect

-LRB-

Fig.

-RRB-

When

all

three

sites

were

removed

there

was

no

induction

by

VegT

and

no

expression

in

the

vegetal

pole

Since

removal

individually

of

either

the

B

Sox

or

the

T-box

sites

but

not

the

A

Sox

site

removed

much

of

the

response

of

maximal

induction

clearly

requires

the

presence

of

both

B

Sox

and

T-box

sites

This

suggests

that

there

is

synergy

between

the

VegT

and

bound

to

these

sites

The

role

of

the

two

Sox

sites

in

responses

to

is

examined

in

Fig.

This

confirms

that

Sox

site

B

has

the

main

activity

and

the

T-box

site

is

unimportant

in

induction

by

alone

It

is

possible

that

in

the

endoderm

itself

the

Sox

sites

respond

to

Sox

proteins

other

than

To

test

this

DNA

was

injected

into

the

vegetal

pole

followed

by

injection

of

the

three

antisense

morpholino

oligos

-LRB-

MOs

-RRB-

that

block

translation

of

the

three

X.

laevis

mRNAs

-LRB-

Clements

et

al.

-RRB-

The

enhanced

activity

of

in

vegetal

poles

as

compared

to

animal

was

eliminated

whereas

a

control

MO

had

no

effect

-LRB-

Fig.

-RRB-

This

confirms

that

the

endogenous

are

responsible

for

the

high

activity

of

the

element

in

the

developing

endoderm

The

element

is

bound

to

in

vivo

To

confirm

that

the

Sox

sites

in

are

functional

we

needed

to

show

that

they

are

occupied

by

in

the

living

embryo

We

therefore

performed

a

chromatin

immunoprecipitation

-LRB-

ChIP

-RRB-

on

mid-gastrulae

stage

embryos

Chromatin

was

isolated

sheared

to

DNA

fragments

of

about

bp

and

immunoprecipitated

with

an

anti-serum

-LRB-

Clements

et

al.

-RRB-

Fig.

shows

PCRs

from

several

regions

in

the

resulting

genomic

DNA

The

plus

element

was

clearly

precipitated

by

the

anti-serum

but

not

pre-immune

serum

Conversely

the

ORF

was

not

precipitated

as

expected

since

it

is

about

kb

distant

from

the

E-element

A

second

control

was

to

probe

for

an

enhancer

previously

used

for

immunoprecipitation

with

an

Xbra

anti-serum

-LRB-

Messenger

et

al.

-RRB-

This

ventrally

expressed

gene

should

not

be

regulated

by

and

indeed

the

promoter

element

fails

to

precipitate

with

antiserum

Therefore

as

expected

from

the

transgenic

data

the

E-element

is

bound

to

in

vivo

Discussion

Expression

of

the

Xenopus

genes

defines

the

endodermal

territory

in

the

mid-blastula

when

cells

start

rapid

transcription

and

make

the

initial

decisions

to

become

endoderm

For

this

reason

we

have

focused

on

the

regulation

of

one

of

these

genes

in

the

blastula

and

early

gastrula

We

have

been

able

to

identify

two

regulatory

elements

at

approximately

minus

kb

that

drive

transcription

in

the

future

endoderm

at

blastula

and

gastrula

stages

Study

of

the

endogenous

genes

has

suggested

that

the

regulation

of

the

genes

is

very

dynamic

passing

rapidly

through

initiation

establishment

and

maintenance

phases

-LRB-

see

Introduction

-RRB-

In

the

initiation

phase

is

a

direct

target

of

the

localised

maternal

transcription

factor

VegT

in

the

establishment

phase

it

becomes

signal-dependent

and

eventually

maintenance

is

cell-autonomous

One

would

hope

to

find

the

basis

of

these

regulatory

processes

in

the

promoter

Activity

of

the

endodermal

element

Of

the

two

adjacent

elements

capable

of

driving

endodermal

expression

responds

strongly

to

VegT

and

and

to

Activin

but

not

to

VegT

We

have

principally

focused

on

but

we

have

made

a

preliminary

analysis

of

This

region

must

contain

cooperating

elements

since

two

fragments

each

containing

half

of

plus

an

overlapping

region

were

negative

in

terms

of

endodermal

expression

The

Activin

responsiveness

of

correlates

with

its

possession

of

three

separate

pairs

of

closely

associated

and

Smad

sites

which

are

known

direct

effectors

of

Activin/Nodal

signalling

-LRB-

Chen

et

al.

Germain

et

al.

-RRB-

However

Activin

induces

the

expression

of

many

downstream

molecules

which

could

then

be

responsible

for

indirect

induction

of

this

element

To

prove

the

effect

was

direct

we

showed

that

is

stimulated

by

activated

in

the

oocyte

This

indicates

a

direct

effect

since

the

single

nucleus

of

this

cell

has

less

transcriptional

capacity

to

induce

downstream

effectors

of

Activin/Smad

action

than

does

the

early

gastrula

Maternal

depletion

of

by

Kofron

et

al.

-LRB-

-RRB-

showed

that

it

was

not

essential

for

expression

However

as

they

point

out

Xenopus

embryos

also

express

which

binds

to

the

same

target

sequence

-LRB-

Howell

et

al.

-RRB-

and

could

therefore

redundantly

regulate

through

the

same

promoter

elements

responds

strongly

to

Activin

but

with

respect

to

vegetal

expression

and

response

to

VegT

its

behaviour

is

paradoxical

It

is

surprising

that

it

does

not

respond

to

VegT

in

the

embryo

both

because

contains

a

consensus

T-box

core

sequence

and

because

VegT

induces

the

expression

of

TGF-βs

that

like

Activin

act

through

Smads

indeed

VegT

depends

on

this

signalling

for

its

overall

biological

effect

The

T-box

site

in

entirely

overlaps

a

pair

of

sites

which

may

have

a

bearing

on

the

fact

that

VegT

actually

inhibits

basal

expression

just

as

its

expression

is

inhibited

in

the

vegetal

pole

compared

to

the

animal

This

is

consistent

with

the

observation

that

removal

of

the

T-box

site

removes

the

inhibition

-LRB-

data

not

shown

-RRB-

was

identified

by

its

ability

to

direct

expression

in

the

vegetal

pole

of

transgenics

however

here

we

simply

scored

expression

in

the

vegetal

pole

itself

disregarding

animal

regions

Conversely

the

luciferase

measurements

are

of

the

ratio

either

of

vegetal

to

animal

expression

or

of

VegT

stimulated

to

control

expression

in

the

animal

region

Therefore

a

high

level

of

animal

expression

would

mask

vegetal

activity

of

the

promoter

However

it

is

important

to

note

that

both

the

VegT

induction

and

vegetal

expression

are

consistent

We

believe

that

the

response

to

TGF-βs

is

the

important

property

of

and

other

problems

are

introduced

by

looking

at

small

regulatory

regions

in

isolation

where

synergising

and

inhibitory

effects

are

absent

Previous

work

has

shown

that

expression

switches

from

direct

VegT

response

to

TGF-β

signal

dependence

is

a

therefore

a

good

candidate

for

controlling

the

signal-dependent

process

It

is

conceivable

that

the

inhibitory

effects

we

notice

bear

on

the

hypothetical

inhibitor

that

make

signal

dependent

in

the

establishment

phase

of

endoderm

development

The

activity

of

the

endodermal

element

The

activity

of

is

more

straightforward

It

is

highly

expressed

in

the

presumptive

endoderm

in

transgenics

and

it

is

much

more

highly

expressed

in

the

vegetal

than

the

animal

hemisphere

in

transient

assays

It

is

also

strongly

stimulated

by

VegT

It

contains

a

divergent

T-box

site

which

binds

VegT

in

vitro

and

which

mutation

shows

is

partially

responsible

for

the

VegT

stimulation

in

the

embryo

Expression

in

oocytes

shows

that

responds

directly

to

VegT

through

this

site

although

the

stimulation

is

less

than

in

embryos

This

correlates

with

the

fact

that

blocking

TGF-β

signalling

in

embryos

with

a

truncated

Activin

receptor

reduces

VegT

stimulation

to

about

the

oocyte

level

Thus

responds

directly

to

VegT

and

synergistically

to

other

molecules

that

are

downstream

of

the

VegT-induced

TGF-βs

which

would

principally

be

Nodal-related

signals

These

synergistic

molecules

are

the

proteins

themselves

in

an

autoregulatory

loop

responds

directly

to

Other

molecules

such

as

Gata

are

downstream

of

and

could

in

principle

regulate

Although

contains

a

possible

variant

Gata-binding

site

mutation

of

the

two

consensus

Sox

sites

removes

much

of

the

activity

of

and

the

triple

Sox/VegT

mutant

is

unresponsive

both

to

VegT

and

vegetal

pole

expression

Further

mutation

of

the

possible

Gata

site

has

no

effect

on

the

response

of

to

VegT

-LRB-

not

shown

-RRB-

The

direct

action

of

on

was

confirmed

by

transcriptional

assays

in

oocytes

These

observations

fit

with

the

fact

that

auto-induces

itself

-LRB-

Sinner

et

al.

-RRB-

but

endogenous

genes

are

very

slow

to

be

induced

when

is

expressed

ectopically

in

the

animal

cap

and

this

is

equally

true

of

the

regulatory

region

Previously

we

have

found

the

same

for

a

direct

Sox

inductive

site

in

the

Endodermin

promoter

-LRB-

Ahmed

et

al.

-RRB-

In

fact

we

believe

that

the

Sox

sites

in

are

already

needed

at

blastula

stages

because

they

are

required

for

full

vegetal

expression

of

the

GFP

transgene

as

early

as

the

onset

to

gastrulation

judged

by

GFP

fluorescence

Since

GFP

takes

several

hours

to

mature

-LRB-

Davis

et

al.

-RRB-

there

must

have

been

an

earlier

requirement

for

the

Sox

factors

We

have

argued

previously

in

connection

with

the

Endodermin

promoter

that

must

normally

co-operate

with

other

molecules

found

in

the

vegetal

pole

In

the

animal

cap

presumably

establishes

the

endodermal

gene

network

more

slowly

than

its

normal

establishment

by

multiple

maternal

inputs

-LRB-

Sinner

et

al.

-RRB-

Since

is

rapidly

induced

by

VegT

and

this

depends

largely

on

the

Sox

sites

and

expression

-LRB-

as

shown

by

Sox

site

mutants

and

blocking

action

with

combined

group

morpholinos

-RRB-

it

seems

that

VegT

is

able

to

co-operate

with

to

produce

more

rapid

Sox

action

The

kinetics

of

reporter

expression

suggest

that

the

VegT

and

the

T-box

site

are

not

simply

needed

at

the

very

onset

of

endodermal

gene

expression

in

the

initiation

phase

but

that

this

continues

into

gastrulation

during

the

establishment

phase

It

is

provocative

that

the

Sox

site

of

principal

importance

in

is

very

near

to

the

VegT

site

suggesting

a

direct

interaction

between

VegT

and

although

we

have

been

unable

to

detect

this

by

immunological

co-precipitation

The

role

of

auto-induction

in

the

endodermal

network

The

idea

that

a

key

endodermal

gene

induces

itself

is

attractive

because

it

would

give

stability

to

expression

and

hence

to

the

endodermal

network

The

requirement

for

signalling

also

adds

a

fail-safe

for

cells

that

become

misplaced

from

the

endodermal

domain

but

still

initiate

direct

VegT-dependent

endodermal

gene

expression

These

conform

to

the

fate

of

their

neighbours

-LRB-

Clements

et

al.

Clements

and

Woodland

Wardle

and

Smith

Wylie

et

al.

Yasuo

and

Lemaire

-RRB-

However

blocking

action

with

morpholino

oligos

has

no

effect

on

expression

even

though

other

targets

including

Endodermin

and

are

down-regulated

-LRB-

Clements

et

al.

Sinner

et

al.

-RRB-

The

most

obvious

reason

for

this

paradox

is

redundancy

For

example

can

be

induced

by

and

and

the

first

two

are

only

partially

and

the

latter

not

at

all

affected

by

morpholinos

-LRB-

Clements

et

al.

and

unpublished

data

-RRB-

is

a

direct

target

-LRB-

Afouda

et

al.

-RRB-

but

while

and

elements

contain

possible

divergent

Gata

sites

mutating

these

in

does

not

prevent

its

response

to

Furthermore

only

the

Sox

and

T-box

sites

of

are

essential

for

expression

in

the

endoderm

While

other

factors

like

Mix/Bix

proteins

could

be

relevant

another

candidate

is

maternal

which

is

localised

in

the

vegetal

pole

-LRB-

Zhang

et

al.

-RRB-

Indeed

we

have

shown

that

overexpression

of

in

the

animal

hemisphere

results

in

the

induction

of

although

again

this

is

slow

as

with

itself

-LRB-

data

not

shown

-RRB-

Nevertheless

the

case

for

auto-regulation

is

compelling

now

being

based

on

three

independent

lines

of

evidence

Firstly

induces

itself

in

ectopic

expression

experiments

and

this

is

direct

-LRB-

Sinner

et

al.

-RRB-

secondly

there

are

essential

Sox

sites

in

a

promoter

element

that

drives

endodermal

gene

expression

in

transient

and

true

transgenics

thirdly

ChIP

analysis

shows

that

the

endodermal

element

is

bound

to

in

vivo

Based

on

the

activity

of

a

glucocorticoid

derivative

of

VegT

in

the

absence

of

protein

synthesis

is

a

direct

target

of

VegT

-LRB-

Clements

and

Woodland

-RRB-

and

our

results

show

that

it

co-operates

with

-LRB-

and

possibly

other

transcription

factors

-RRB-

to

establish

and

amplify

gene

expression

The

fact

that

expression

is

ultimately

dependent

on

TGF-β

signalling

makes

this

amplification

and

stabilisation

of

its

own

expression

signal-sensitive

and

hence

subject

to

a

community

effect

-LRB-

Gurdon

et

al.

b

-RRB-

We

propose

that

this

involves

the

element

A

simplified

version

of

this

network

is

shown

in

Fig.

An

important

point

to

note

is

that

other

members

of

the

endodermal

network

like

the

Mix/Bix

genes

are

similar

to

with

respect

to

VegT

induction

and

signal

dependence

There

are

still

many

issues

to

be

resolved

relating

to

the

regulatory

elements

A

VegT-induced

inhibitor

of

its

own

action

in

inducing

was

postulated

to

explain

the

switch

to

signal

dependence

of

VegT

action

and

this

is

still

unknown

It

may

relate

to

the

repressive

effect

of

VegT

and

vegetal

position

on

a

repression

that

is

lifted

by

TGF-β

signalling

There

also

appear

to

be

inhibitors

of

expression

in

other

parts

of

the

embryo

For

example

the

loss

of

the

Sox

sites

appears

to

increase

the

background

expression

in

the

animal

pole

This

could

involve

the

action

of

the

inhibitor

-LRB-

Zhang

et

al.

-RRB-

At

later

stages

there

is

also

expression

of

most

transgenes

in

regions

like

the

axis

where

is

not

expressed

This

was

not

seen

in

a

construct

including

kb

of

sequence

suggesting

that

late

inhibitory

sites

are

present

in

this

region

The

regulatory

interactions

described

here

are

elements

within

the

core

endodermal

network

-LRB-

Sinner

et

al.

-RRB-

The

form

of

the

regulation

where

VegT

induces

several

factors

such

as

TGF-βs

and

and

co-operates

with

them

to

regulate

an

important

control

gene

is

an

important

recurring

regulatory

motif

the

feed-forward

motif

-LRB-

Mangan

and

Alon

-RRB-

In

this

case

it

also

brings

about

autoregulation

Autoregulatory

effects

of

proteins

in

transcription

factor

networks

are

thought

to

have

special

importance

Mochizuki

-LRB-

-RRB-

found

that

the

number

of

possible

stable

states

of

gene

networks

depended

only

on

the

number

of

autoregulated

components

Since

it

is

reasonable

to

equate

stable

states

with

differentiated

states

the

autoregulation

of

a

key

endodermal

transcription

factor

would

be

most

significant

in

endodermal

differentiation

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