Do

teashirt

family

genes

specify

trunk

identity

Insights

from

the

single

tiptop/teashirt

homolog

of

Tribolium

castaneum

The

Drosophila

teashirt

gene

acts

in

concert

with

the

homeotic

selector

-LRB-

Hox

-RRB-

genes

to

specify

trunk

-LRB-

thorax

and

abdomen

-RRB-

identity

There

has

been

speculation

that

this

trunk-specifying

function

might

be

very

ancient

dating

back

to

the

common

ancestor

of

insects

and

vertebrates

However

other

evidence

suggests

that

the

role

of

teashirt

in

trunk

identity

is

not

well

conserved

even

within

the

Insecta

To

address

this

issue

we

have

analyzed

the

function

of

Tc-tiotsh

the

lone

teashirt

family

member

in

the

red

flour

beetle

Tribolium

castaneum

Although

Tc-tiotsh

is

important

for

aspects

of

both

embryonic

and

imaginal

development

including

some

trunk

features

we

find

no

evidence

that

it

acts

as

a

trunk

identity

gene

We

discuss

this

finding

in

the

context

of

recent

insights

into

the

evolution

and

function

of

the

Drosophila

teashirt

family

genes

Introduction

Homeotic

genes

are

responsible

for

defining

regional

identity

and

mutations

in

these

genes

often

transform

one

body

part

into

another

The

best

characterized

homeotic

genes

are

the

Hox

genes

which

have

been

identified

in

representatives

of

most

animal

phyla

-LRB-

reviewed

by

Martindale

-RRB-

and

have

been

extensively

studied

in

several

organisms

However

some

non-Hox

genes

have

also

been

classified

as

homeotic

genes

based

on

their

mutant

phenotypes

One

such

gene

encodes

the

zinc

finger

transcription

factor

Teashirt

-LRB-

Fasano

et

al.

-RRB-

Loss-of-function

-LRB-

LOF

-RRB-

teashirt

-LRB-

tsh

-RRB-

mutations

are

reported

to

cause

partial

transformation

of

the

larval

prothorax

to

a

labial

identity

due

to

ectopic

expression

of

the

Hox

gene

Sex

combs

reduced

-LRB-

Fasano

et

al.

Roder

et

al.

-RRB-

The

trunk

-LRB-

thoracic

and

abdominal

-RRB-

segments

of

tsh

larvae

are

shorter

than

normal

and

the

denticle

belts

-LRB-

a

trunk-specific

feature

-RRB-

are

severely

disorganized

Moreover

patches

of

sclerotic

cuticle

which

have

been

interpreted

as

head

structures

form

between

the

remaining

denticle

belts

-LRB-

Fasano

et

al.

-RRB-

Conversely

ectopic

expression

of

tsh

results

in

the

development

of

denticle

belts

in

the

head

-LRB-

de

Zulueta

et

al.

-RRB-

On

the

basis

of

these

phenotypes

tsh

has

been

suggested

to

function

as

a

specifier

of

trunk

vs

head

identity

-LRB-

Roder

et

al.

-RRB-

tsh

is

believed

to

act

in

parallel

with

the

trunk

Hox

genes

-LRB-

Scr

Antennapedia

-LRB-

Antp

-RRB-

Ultrabithorax

-LRB-

Ubx

-RRB-

abdominal-A

-LRB-

abd-A

-RRB-

and

Abdominal-B

-LRB-

Abd-B

-RRB-

-RRB-

to

determine

segmental

identity

-LRB-

de

Zulueta

et

al.

Roder

et

al.

-RRB-

When

all

of

these

Hox

genes

are

inactivated

trunk

segments

develop

with

what

has

been

interpreted

as

a

mixed

head/trunk

identity

That

is

sclerotized

tissue

develops

in

the

posterior

compartment

of

each

trunk

segment

whereas

denticle

belts

-LRB-

a

trunk-specific

feature

-RRB-

still

develop

in

the

anterior

compartment

-LRB-

Sato

et

al.

Struhl

-RRB-

When

tsh

function

is

also

eliminated

the

trunk

segments

are

more

severely

affected

in

that

denticle

belts

disappear

and

additional

head-like

cuticle

is

observed

-LRB-

Roder

et

al.

-RRB-

Recent

data

suggests

that

tsh

may

play

an

even

greater

role

in

trunk

development

than

was

previously

recognized

In

the

absence

of

tsh

function

its

paralog

tiptop

-LRB-

tio

-RRB-

is

ectopically

expressed

in

the

trunk

region

-LRB-

Laugier

et

al.

-RRB-

When

tio

and

tsh

functions

are

both

absent

the

trunk

denticles

are

even

more

severely

affected

than

in

mutants

that

lack

only

tsh

suggesting

that

tio

can

partially

substitute

for

tsh

and

reduce

the

effects

of

tsh

mutations

Robertson

and

coworkers

-LRB-

-RRB-

proposed

the

existence

of

a

system

of

zinc-finger

proteins

that

divides

the

Drosophila

embryo

into

sections

and

limits

the

functional

domains

of

Hox

genes

They

suggest

that

within

this

system

tsh

specifies

the

trunk

region

while

disconnected

-LRB-

disco

-RRB-

specifies

the

gnathal

region

In

the

absence

of

tsh

disco

-LRB-

as

well

as

its

paralog

disco-related

-RRB-

is

ectopically

expressed

in

the

trunk

and

there

is

some

evidence

that

ectopic

disco

expression

is

responsible

for

at

least

some

features

of

tsh

mutants

-LRB-

e.g.

loss

of

denticles

and

abnormal

sensory

neurons

-RRB-

that

are

thought

to

represent

a

switch

to

head/gnathal

identity

-LRB-

Robertson

et

al.

-RRB-

Manfroid

et

al.

-LRB-

-RRB-

speculated

that

the

trunk-specifying

function

of

tsh

family

members

might

be

conserved

in

vertebrates

as

tsh

genes

from

mice

are

expressed

in

the

trunk

region

and

can

also

rescue

the

phenotype

of

Drosophila

tsh

mutants

when

ectopically

expressed

However

such

conjecture

requires

a

rather

large

phylogenetic

leap

as

it

is

not

clear

that

the

role

of

tsh

in

trunk

specification

is

conserved

even

in

other

insects

tsh

family

genes

of

the

firebrat

Thermobia

domestica

and

the

milkweed

bug

Oncopeltus

fasciatus

are

expressed

predominantly

in

the

gnathos

and

thorax

arguing

against

a

function

in

trunk

specification

-LRB-

Herke

et

al.

Peterson

et

al.

-RRB-

Furthermore

when

Herke

et

al.

-LRB-

-RRB-

knocked

down

the

function

of

the

Oncopeltus

tsh

family

gene

by

RNA

interference

-LRB-

RNAi

-RRB-

they

observed

transformations

of

distal

leg

to

antenna

but

did

not

report

any

changes

in

trunk

identity

Finally

our

previous

studies

suggest

that

genetic

specification

of

trunk

identity

in

the

red

flour

beetle

Tribolium

castaneum

differs

significantly

from

that

in

Drosophila

Removal

of

trunk

Hox

genes

alone

is

sufficient

to

produce

dramatic

transformation

of

trunk

to

head

identity

-LRB-

Brown

et

al.

-RRB-

Antennae

develop

on

all

gnathal

and

trunk

segments

of

embryos

homozygous

for

a

deficiency

that

removes

the

Tribolium

orthologs

of

Deformed

-LRB-

Dfd

-RRB-

Scr

Antp

Ubx

and

abd-A

-LRB-

Stuart

et

al.

-RRB-

Thus

there

is

apparently

no

need

to

invoke

the

function

of

another

gene

for

trunk

identity

Peterson

et

al.

-LRB-

-RRB-

predicted

that

either

the

Tribolium

tsh

gene

has

no

function

in

trunk

specification

or

that

the

expression

of

tsh

is

controlled

by

Hox

genes

Taken

together

these

observations

suggest

that

the

trunk-specifying

role

of

tsh

observed

in

Drosophila

might

not

be

conserved

in

other

insects

To

better

understand

the

evolution

of

tsh

family

gene

function

we

cloned

and

characterized

a

tsh

homolog

from

Tribolium

This

gene

Tc-tiotsh

is

the

sole

member

of

this

family

in

the

Tribolium

genome

It

has

a

broad

embryonic

expression

domain

encompassing

not

only

the

trunk

region

but

much

of

the

head

as

well

RNAi

experiments

revealed

that

Tc-tiotsh

functions

in

several

embryonic

and

imaginal

tissues

but

they

did

not

suggest

a

conserved

function

in

trunk

specification

We

discuss

these

results

and

reexamine

the

classification

of

tsh

as

a

trunk-specifying

gene

in

Drosophila

Materials

and

methods

cDNA

cloning

A

genomic

fragment

of

the

Tc-tiotsh

gene

was

amplified

using

degenerate

primers

based

on

the

conserved

amino

acid

motifs

VWLGKG

and

CKVCDK

-LRB-

first

round

-RRB-

and

ILKCMW

and

CKVCDK

-LRB-

second

round

-RRB-

Additional

overlapping

fragments

were

identified

by

RACE

using

the

and

RACE

Systems

-LRB-

GibcoBRL

-RRB-

by

polymerase

chain

reaction

-LRB-

PCR

-RRB-

screening

a

Tribolium

castaneum

cDNA

library

as

described

previously

-LRB-

Nie

et

al.

-RRB-

or

by

inverse

PCR

-LRB-

Ochman

et

al.

-RRB-

To

confirm

that

these

overlapping

fragments

represent

a

contiguous

sequence

primers

were

designed

near

the

ends

of

the

putative

contig

and

reverse

transcriptase

-LRB-

RT

-RRB-

PCR

was

performed

using

total

embryonic

RNA

-LRB-

h

of

development

-RRB-

as

template

The

resulting

PCR

product

was

cloned

and

sequenced

-LRB-

GenBank

accession

number

-RRB-

Sequence

analysis

The

nucleotide

contig

for

Tc-tiotsh

was

assembled

using

the

Seqman

module

of

Lasergene

-LRB-

DNASTAR

-RRB-

To

identify

putative

tsh

family

members

in

other

genomes

we

performed

tBLASTn

searches

using

Drosophila

melanogaster

Tsh

and

Tio

as

query

sequences

Multiple

alignments

were

created

with

the

AlignX

module

of

Vector

NTI

-LRB-

InforMax

-RRB-

Minor

manual

adjustments

were

performed

to

maximize

the

alignments

The

final

output

of

the

multiple

alignment

was

created

using

BOXSHADE

-LRB-

available

at

-RRB-

Phylogenetic

analyses

were

performed

using

MEGA

version

-LRB-

Kumar

et

al.

-RRB-

or

TreePuzzle

-LRB-

-RRB-

and

TreeView

-LRB-

-RRB-

was

used

to

visualize

the

output

In

situ

hybridization

and

immunostaining

of

embryos

A

digoxigenin-labeled

fragment

of

Tc-tiotsh

was

hybridized

to

whole-mount

wild-type

-LRB-

-RRB-

embryos

as

previously

described

-LRB-

Brown

et

al.

-RRB-

Embryos

were

secondarily

immunostained

with

either

α-Invected

antibody

-LRB-

-RRB-

-LRB-

Patel

et

al.

-RRB-

or

with

α-Even-skipped

antibody

-LRB-

-RRB-

-LRB-

Patel

et

al.

-RRB-

which

cross-react

with

Tc-Engrailed

and

Tc-Even-skipped

respectively

Tc-spalt

-LRB-

Tc-sal

-RRB-

expression

was

assayed

using

the

probe

described

by

Tomoyasu

et

al.

-LRB-

-RRB-

RNA

interference

-LRB-

RNAi

-RRB-

dsRNA

of

two

different

non-overlapping

fragments

of

Tc-tiotsh

was

synthesized

using

the

PCR

template

method

described

by

Kennerdell

and

Carthew

-LRB-

-RRB-

Briefly

primers

were

chosen

to

amplify

a

fragment

of

Tc-tiotsh

-LRB-

bases

or

of

GenBank

sequence

-RRB-

and

the

promoter

sequence

-LRB-

TAATACGACTCACTATAGG

-RRB-

was

added

to

the

end

of

each

primer

The

resulting

primers

were

used

to

amplify

template

for

RNA

synthesis

The

resulting

product

was

purified

and

μg

used

as

template

for

RNA

synthesis

using

the

Megascript

High

Yield

Transcription

kit

-LRB-

Ambion

-RRB-

For

embryonic

RNAi

h

eggs

were

collected

from

wild-type

beetles

-LRB-

-RRB-

and

injected

with

dsRNA

-LRB-

μg/μl

-RRB-

as

previously

described

by

Brown

et

al.

-LRB-

-RRB-

After

several

days

hatched

larvae

and

unhatched

eggs

were

treated

with

lactic

acid/ethanol

Additional

larvae

were

documented

by

SEM

as

described

by

Curtis

et

al.

-LRB-

-RRB-

Injection

into

non-virgin

adult

females

was

performed

by

manually

opening

the

elytra

and

wings

of

ether-anesthetized

beetles

and

injecting

μg/μl

dsRNA

into

the

dorsal

abdomen

Larval

RNAi

was

performed

as

described

by

Tomoyasu

and

Denell

-LRB-

-RRB-

Results

Tribolium

has

a

single

teashirt

family

gene

We

cloned

a

member

of

the

tsh

gene

family

from

the

red

flour

beetle

Tribolium

castaneum

-LRB-

see

Materials

and

methods

for

details

-RRB-

Comparison

of

the

cDNA

sequence

to

the

recently

completed

Tribolium

genome

sequence

revealed

three

exons

distributed

over

about

kb

-LRB-

Fig.

-RRB-

Most

of

this

distance

can

be

attributed

to

an

approximately

intron

between

the

first

and

second

exons

Fig.

structure

of

the

Tc-tiotsh

gene

and

its

Drosophila

homologs

Exons

are

denoted

by

boxes

and

introns

by

thin

lines

The

coding

region

is

shaded

gray

and

zinc

finger-encoding

motifs

are

shown

in

black

Only

three

full-length

Tsh

family

proteins

have

been

described

from

insects

the

Drosophila

melanogaster

paralogs

Tsh

and

Tio

and

the

single

member

of

the

Tsh

family

in

Anopheles

gambiae

Ag-TioTsh

-LRB-

Fasano

et

al.

Laugier

et

al.

-RRB-

In

addition

small

fragments

of

tsh

family

genes

have

been

amplified

from

several

other

insects

-LRB-

Herke

et

al.

Peterson

et

al.

-RRB-

Two

conclusions

have

been

drawn

from

these

limited

data

First

the

two

Drosophila

genes

seem

to

be

the

result

of

a

recent

duplication

in

the

Drosophila

lineage

Second

all

of

the

Tsh

family

members

from

other

insects

appear

to

be

more

similar

in

sequence

to

Tio

than

to

Tsh

In

this

paper

we

present

data

that

support

and

greatly

strengthen

these

conclusions

We

performed

a

tBLASTn

search

of

several

recently

sequenced

insect

genomes

for

tsh

family

members

-LRB-

Table

-RRB-

The

Tribolium

castaneum

gene

that

we

cloned

is

the

only

member

of

the

tsh

family

present

in

the

genome

sequence

of

this

insect

Likewise

the

Apis

mellifera

-LRB-

honeybee

-RRB-

genome

contains

only

a

single

member

of

this

gene

family

In

contrast

all

of

the

additional

Drosophila

species

we

searched

-LRB-

D.

pseudoobscura

D.

simulans

D.

yakuba

and

D.

virilis

-RRB-

have

orthologs

of

both

tio

and

tsh

Consistent

with

the

situation

in

D.

melanogaster

-LRB-

Laugier

et

al

-RRB-

tio

and

tsh

are

closely

linked

-LRB-

less

than

kb

apart

-RRB-

in

all

of

the

Drosophila

species

examined

suggesting

that

these

genes

likely

arose

from

a

relatively

recent

tandem

duplication

event

Table

family

genes

in

sequenced

insect

genomesOrganismOrdertsh

family

genesGenome

ReferenceTribolium

Tribolium

Genome

Sequencing

Consortium

Honeybee

Genome

Sequencing

Consortium

et

al.

-LRB-

tio

and

tsh

-RRB-

Drosophila

Genomes

Consortium

-LRB-

tio

and

tsh

-RRB-

Drosophila

Genomes

Consortium

-LRB-

tio

and

tsh

-RRB-

Richards

et

al.

-LRB-

tio

and

tsh

-RRB-

Drosophila

Genomes

Consortium

searches

were

used

to

determine

the

number

of

teashirt

-LRB-

tsh

-RRB-

family

genes

in

several

assembled

insect

genomes

All

Drosophila

species

examined

had

orthologs

of

both

tsh

and

tiptop

-LRB-

tio

-RRB-

while

the

non-drosophilid

species

each

contained

only

one

tsh

family

member

As

the

Tribolium

gene

is

related

to

both

tio

and

tsh

we

have

named

it

Tc-tiotsh

after

the

convention

established

by

Laugier

et

al.

-LRB-

-RRB-

for

the

single

Anopheles

gene

We

compared

the

predicted

Tc-TioTsh

protein

to

the

other

insect

Tsh

family

members

for

which

full-length

predicted

proteins

are

available

-LRB-

Although

we

were

able

to

identify

a

tsh

family

gene

in

Apis

the

portion

of

the

transcription

unit

was

apparently

missing

from

the

genome

sequence

-RRB-

Like

tio

and

Ag-tiotsh

-LRB-

Laugier

et

al.

-RRB-

Tc-tiotsh

encodes

four

zinc

fingers

-LRB-

Fig.

-RRB-

three

of

which

are

clustered

near

the

middle

of

the

protein

-LRB-

Fig.

-RRB-

and

one

that

is

located

near

the

C

terminus

-LRB-

Fig.

-RRB-

Besides

the

zinc

finger

regions

several

additional

short

regions

of

sequence

identity

-LRB-

shaded

in

Fig.

and

data

not

shown

-RRB-

are

present

in

all

of

the

full-length

predicted

proteins

The

three

dipteran

proteins

are

highly

similar

at

their

N

termini

while

Tio

Ag-TioTsh

and

Tc-TioTsh

share

a

C-terminal

motif

-LRB-

in

addition

to

the

fourth

zinc

finger

-RRB-

that

is

absent

in

Tsh

Regions

shared

between

the

beetle

and

dipteran

proteins

have

been

conserved

over

a

long

phylogenetic

distance

suggesting

that

they

likely

have

important

functions

Fig.

alignments

of

Teashirt

family

members

Amino

acids

identical

or

similar

in

at

least

two

of

the

proteins

are

shaded

black

or

gray

respectively

Tc

Tribolium

castaneum

Ag

Anopheles

gambiae

Dm

Drosophila

melanogaster

Mm

Mus

musculus

Dr

Danio

rerio

a

Multiple

alignment

of

the

three

clustered

zinc

fingers

Zinc

fingers

-LRB-

ZF

-RRB-

are

indicated

above

the

sequence

Regions

underscored

with

asterisks

were

concatenated

and

subjected

to

phylogenetic

analysis

b

Multiple

alignment

of

the

C-terminal

zinc

finger

region

Zinc

finger

is

delimited

above

the

alignment

Tsh

lacks

this

zinc

finger

c

Distinct

N-terminal

motifs

in

dipteran

Tsh

family

members

-LRB-

upper

-RRB-

and

in

Tribolium

and

vertebrates

-LRB-

lower

-RRB-

To

better

understand

the

evolutionary

history

of

the

tsh

gene

family

we

have

performed

phylogenetic

analyses

using

conserved

portions

of

the

zinc-finger

region

-LRB-

underlined

in

Fig.

-RRB-

from

the

full-length

insect

Tsh

family

proteins

that

are

currently

available

-LRB-

Drosophila

Tribolium

and

Anopheles

-RRB-

Both

maximum

likelihood

-LRB-

Fig.

-RRB-

and

neighbor-joining

-LRB-

data

not

shown

-RRB-

methods

produce

trees

in

which

the

two

Drosophila

proteins

cluster

together

suggesting

these

genes

were

produced

by

duplication

after

the

divergence

of

the

mosquito

and

fly

lineages

The

branch

lengths

of

the

Drosophila

proteins

are

much

longer

than

those

of

Anopheles

and

Tribolium

suggesting

rapid

divergence

of

both

genes

after

duplication

Fig.

analysis

of

insect

Teashirt

family

members

The

two

Drosophila

paralogs

cluster

together

in

an

unrooted

tree

produced

by

maximum

likelihood

analysis

suggesting

that

these

genes

resulted

from

a

recent

duplication

in

the

Drosophila

lineage

The

bootstrap

value

of

indicates

that

the

tree

is

very

robust

The

mouse

and

human

genomes

each

have

three

tsh

family

members

-LRB-

Caubit

et

al.

Manfroid

et

al.

-RRB-

which

are

much

more

similar

to

one

another

than

they

are

to

any

of

the

insect

proteins

Interestingly

vertebrate

Tsh

family

proteins

share

an

N-terminal

motif

-LRB-

MPRRKQ

-RRB-

with

the

Tribolium

protein

-LRB-

Fig.

-RRB-

which

is

distinct

from

the

N-terminal

motif

shared

by

the

dipteran

family

members

-LRB-

Laugier

et

al.

-RRB-

The

conservation

of

this

sequence

in

an

insect

and

vertebrates

suggests

it

has

an

important

function

and

that

it

was

probably

present

in

the

common

ancestor

of

protostomes

and

deuterostomes

Taken

together

these

comparisons

of

tsh

family

members

indicate

that

the

ancestor

of

protostomes

and

deuterostomes

had

a

single

tsh

family

gene

We

were

unable

to

identify

any

tsh

family

members

in

the

genomes

of

two

non-bilaterian

metazoans

the

placozoan

Trichoplax

adhaerens

-LRB-

-RRB-

and

the

cnidarian

Nematostella

vectensis

-LRB-

Putnam

et

al.

-RRB-

suggesting

this

gene

family

may

have

arisen

after

the

bilaterian/non-bilaterian

split

Within

the

vertebrate

lineage

duplication

events

-LRB-

perhaps

in

association

with

whole

genome

duplications

-RRB-

produced

at

least

three

paralogs

A

duplication

in

the

Drosophila

lineage

-LRB-

after

divergence

from

the

mosquito

lineage

-RRB-

resulted

in

the

tsh

and

tio

paralogs

Tc-tiotsh

embryonic

expression

is

not

limited

to

the

trunk

We

examined

the

embryonic

expression

pattern

of

Tc-tiotsh

-LRB-

Fig.

-RRB-

by

in

situ

hybridization

using

the

posterior

compartment

marker

Engrailed

-LRB-

Brown

et

al.

Patel

et

al.

-RRB-

to

determine

the

stage

of

embryonic

development

-LRB-

segments

are

added

sequentially

-RRB-

and

provide

a

reference

for

the

register

of

Tc-tiotsh

expression

Tc-tiotsh

expression

develops

in

a

dynamic

pair

rule-like

manner

-LRB-

Fig

and

-RRB-

but

eventually

encompasses

not

only

the

trunk

segments

-LRB-

thorax

and

abdomen

-RRB-

but

also

the

gnathal

segments

and

the

labrum

and

intercalary

segment

in

the

procephalon

-LRB-

Fig.

-RRB-

Tc-tiotsh

expression

in

the

thorax

and

the

first

abdominal

segment

appears

somewhat

stronger

than

in

other

regions

-LRB-

Fig.

-RRB-

As

appendages

develop

Tc-tiotsh

is

expressed

in

the

proximal

portion

of

each

leg

-LRB-

Fig.

-RRB-

Fig.

expression

in

Tribolium

embryos

Engrailed

expression

is

shown

in

brown

while

Tc-tiotsh

expression

is

in

purple

Embryos

are

oriented

with

anterior

to

the

left

-LRB-

except

in

panel

d

in

which

anterior

is

to

the

top

-RRB-

a

During

germband

elongation

Tc-tiotsh

is

expressed

in

the

gnathal

and

trunk

segments

as

well

as

two

procephalic

regions

-LRB-

the

intercalary

segment

-LSB-

Ic

-RSB-

and

the

labrum

-LSB-

Lr

-RSB-

-RRB-

b

When

germband

elongation

is

completed

Tc-tiotsh

expression

is

strongest

in

the

thoracic

-LRB-

-RRB-

and

first

abdominal

-LRB-

-RRB-

segments

c

Slightly

later

strong

Tc-tiotsh

expression

appears

in

the

posterior

terminal

region

of

the

embryo

-LRB-

arrowhead

-RRB-

dTc-tiotsh

is

expressed

in

only

the

proximal

portion

-LRB-

arrow

-RRB-

of

the

developing

legs

e

In

embryos

undergoing

germ-band

retraction

Tc-tiotsh

expression

is

present

in

the

six

Malpighian

tubule

primordia

that

evert

from

the

hindgut

f

In

a

Df

-LRB-

HOMC

-RRB-

homozygote

lacking

most

Hox

genes

Tc-tiotsh

expression

in

the

segmented

portion

of

the

germband

is

greatly

reduced

In

contrast

expression

in

the

Malpighian

tubule

primordia

remains

strong

After

germ-band

elongation

Tc-tiotsh

is

expressed

in

the

posterior

region

of

the

embryo

Initially

expression

is

seen

in

a

patch

on

either

side

of

the

ventral

midline

-LRB-

Fig.

-RRB-

and

later

in

six

buds

that

extend

from

the

invaginating

hindgut

-LRB-

Fig.

-RRB-

These

structures

are

the

primordia

of

the

Malpighian

tubules

they

also

express

Tc-dachsund

-LRB-

Prpic

et

al.

-RRB-

and

Tc-Cut

-LRB-

data

not

shown

-RRB-

spalt

does

not

appear

to

repress

Tc-tiotsh

in

Tribolium

embryos

In

Drosophila

the

limits

of

tsh

expression

are

controlled

either

directly

or

indirectly

by

spalt-major

-LRB-

salm

-RRB-

In

a

salm

mutant

the

tsh

expression

domain

expands

both

anteriorly

and

posteriorly

-LRB-

Roder

et

al.

-RRB-

To

assess

whether

the

broader

expression

of

Tc-tiotsh

-LRB-

compared

to

tsh

-RRB-

might

reflect

a

difference

in

Tribolium

spalt

-LRB-

Tc-sal

-RRB-

expression

we

examined

the

embryonic

expression

pattern

of

the

single

Tribolium

sal

gene

-LRB-

Tomoyasu

et

al.

-RRB-

We

find

that

the

Tc-sal

expression

domain

-LRB-

Fig

-RRB-

significantly

overlaps

that

of

Tc-tiotsh

suggesting

that

Tc-sal

does

not

repress

Tc-tiotsh

expression

It

is

possible

that

Tribolium

has

lost

the

regulatory

interaction

between

these

genes

Alternatively

regulation

of

tsh

by

sal

may

have

evolved

more

recently

after

the

divergence

of

these

lineages

Tc-tiotsh

expression

is

reduced

in

embryos

lacking

Hox

genes

In

Drosophila

some

aspects

of

tsh

expression

are

regulated

by

the

Hox

genes

-LRB-

Roder

et

al.

-RRB-

We

have

examined

expression

of

Tc-tiotsh

in

embryos

homozygous

for

Df

-LRB-

HOMC

-RRB-

a

deletion

that

removes

most

of

the

Tribolium

Hox

cluster

Tc-tiotsh

expression

is

severely

reduced

in

the

ventral

region

of

these

embryos

-LRB-

Fig.

-RRB-

These

results

suggest

that

reduction

of

Tc-tiotsh

expression

might

contribute

to

the

transformations

seen

in

Df

-LRB-

HOMC

-RRB-

embryos

However

our

functional

studies

of

Tc-tiotsh

-LRB-

presented

below

-RRB-

reveal

no

evidence

for

this

Tc-tiotsh

functions

in

the

embryonic

labial

and

thoracic

segments

We

used

RNAi

to

ascertain

the

probable

LOF

phenotype

of

Tc-tiotsh

Pupae

injected

with

Tc-tiotsh

dsRNA

produced

very

few

eggs

upon

reaching

adulthood

and

of

these

progeny

showed

an

abnormal

phenotype

To

circumvent

the

apparent

effect

of

Tc-tiotsh

on

oogenesis

we

injected

Tc-tiotsh

dsRNA

either

directly

into

eggs

or

into

the

abdomens

of

non-virgin

adult

females

-LRB-

egg

production

from

these

females

quickly

tapers

off

further

indicating

an

effect

on

oogenesis

-RRB-

Injection

of

Tc-tiotsh

dsRNA

by

either

technique

produced

embryos

with

defects

in

the

labial

and

thoracic

segments

-LRB-

Fig.

Table

and

data

not

shown

-RRB-

During

normal

Tribolium

embryonic

development

the

two

labial

appendages

fuse

at

the

ventral

midline

and

migrate

anteriorly

to

a

position

between

the

maxillary

appendages

-LRB-

Fig.

-RRB-

In

moderately

affected

Tc-tiotsh

RNAi

larvae

-LRB-

Fig.

-RRB-

the

labial

appendages

are

smaller

than

normal

and

in

the

most

severely

affected

embryos

-LRB-

Fig.

-RRB-

these

appendages

fail

to

fuse

In

all

of

these

cases

the

abnormal

labial

appendages

fail

to

migrate

to

their

normal

position

between

the

maxillary

palps

These

abnormalities

produce

secondary

effects

on

the

orientation

of

the

other

head

appendages

Fig.

electron

micrographs

of

Tc-tiotsh

RNAi

larvae

For

all

panels

the

scale

bar

represents

a

c

Ventral

views

of

larval

heads

Maxillary

-LRB-

pink

-RRB-

and

labial

-LRB-

green

-RRB-

appendages

have

been

shaded

d

e

Lateral

views

with

leg

segments

labeled

as

follows

coxa

-LRB-

C

-RRB-

trochanter

-LRB-

Tr

-RRB-

femur

-LRB-

F

-RRB-

tibiotarsus

-LRB-

Tt

-RRB-

and

pretarsus

-LRB-

Pt

-RRB-

a

Wild-type

larval

head

showing

the

proper

position

of

the

maxillary

-LRB-

Mx

-RRB-

and

labial

-LRB-

L

-RRB-

appendages

b

Moderately

affected

Tc-tiotsh

RNAi

larva

in

which

the

labial

appendages

-LRB-

especially

the

proximal

region

-RRB-

are

highly

reduced

c

Severely

affected

Tc-tiotsh

RNAi

larva

in

which

the

labial

appendages

have

failed

to

fuse

Note

that

in

both

b

and

c

the

ventral

posterior

region

of

the

head

-LRB-

presumed

to

derive

from

the

labial

segment

-RRB-

is

almost

completely

absent

d

Wild-type

larva

eTc-tiotsh

RNAi

larva

with

short

legs

On

each

leg

the

trochanter

is

presumably

fused

with

the

reduced

coxaTable

RNA

interference

cuticular

phenotypesdsRNA

fragmentSeverity

of

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

All

results

were

obtained

by

injection

of

dsRNA

into

eggs

dsRNA

fragment

names

refer

to

the

nucleotides

of

GenBank

sequence

included

in

the

fragment

Severity

of

effect

was

classified

as

follows

Larvae

with

mild

effects

had

short

legs

resulting

from

reduction

and

fusion

of

proximal

segments

Moderately

affected

larvae

had

reductions

in

the

ventral

head

and

labial

appendages

in

addition

to

short

legs

Severely

affected

larvae

were

distinguished

from

those

with

moderate

effects

by

the

failure

of

the

labial

appendages

to

fuse

at

the

ventral

midline

Short

legs

were

the

most

common

feature

seen

in

Tc-tiotsh

RNAi

larvae

being

present

in

all

larvae

with

labial

defects

as

well

as

in

some

larvae

with

apparently

normal

labial

appendages

-LRB-

Table

-RRB-

The

reduction

in

leg

length

stems

from

effects

on

the

proximal

leg

segments

The

coxa

and

trochanter

are

reduced

and

apparently

fused

and

the

femur

is

slightly

shortened

-LRB-

Fig.

-RRB-

This

phenotype

is

similar

to

that

observed

in

the

adult

legs

of

hypomorphic

Drosophila

tsh

mutants

-LRB-

Erkner

et

al.

-RRB-

and

thus

may

indicate

a

conserved

role

for

tsh

family

genes

in

proximal

limb

development

We

also

noted

that

Tc-tiotsh

RNAi

larvae

show

a

pronounced

curvature

of

the

head

and

thoracic

segments

such

that

the

head

bends

forward

-LRB-

Fig.

-RRB-

This

phenotype

apparently

results

from

reduction

in

length

-LRB-

anterior

to

posterior

-RRB-

of

the

ventral

portions

of

the

thoracic

segments

and

posterior

head

-LRB-

Fig.

c

f

-RRB-

Tc-tiotsh

is

important

for

proper

development

of

adult

trunk

segments

We

examined

the

role

of

Tc-tiotsh

in

adult

development

using

larval

RNAi

-LRB-

Tomoyasu

and

Denell

-RRB-

The

resulting

adults

display

a

number

of

defects

in

the

thoracic

and

abdominal

segments

-LRB-

Fig.

-RRB-

Most

noticeably

these

individuals

have

abnormalities

of

the

notum

that

prevent

the

elytra

from

closing

properly

-LRB-

data

not

shown

-RRB-

We

also

observed

mild

defects

in

the

proximal

legs

In

addition

adults

resulting

from

Tc-tiotsh

larval

RNAi

show

abnormalities

in

many

of

the

ventral

sclerites

-LRB-

sternites

-RRB-

-LRB-

see

El-Kifl

for

a

description

of

wild-type

structures

-RRB-

In

the

ventral

prothorax

-LRB-

-RRB-

of

RNAi-treated

individuals

the

epimera

which

are

normally

located

posterior

to

the

coxae

are

missing

and

the

basisternum

is

reduced

in

size

resulting

in

the

exposure

of

membranous

areas

-LRB-

Fig.

d

-RRB-

In

addition

the

shape

of

the

sternellum

-LRB-

the

process

extending

between

the

legs

-RRB-

is

abnormal

Ventral

is

not

noticeably

affected

but

the

sternite

is

missing

its

anterior

process

-LRB-

meso-spinasternum

-RRB-

-LRB-

Fig.

-RRB-

The

anterior

abdominal

segments

are

also

affected

by

Tc-tiotsh

larval

RNAi

In

wild-type

adults

the

first

and

second

abdominal

segments

-LRB-

-RRB-

form

an

internal

portion

of

the

leg

sockets

and

are

only

lightly

sclerotized

while

is

distinguished

by

a

sharply

pointed

process

extending

between

the

legs

-LRB-

Fig.

-RRB-

In

Tc-tiotsh

RNAi

individuals

an

additional

anterior

region

-LRB-

and/or

-RRB-

is

heavily

sclerotized

and

lacks

its

anterior

process

thus

resembling

the

more

posterior

abdominal

segments

-LRB-

-RRB-

-LRB-

Fig.

-RRB-

Fig.

phenotypes

resulting

from

Tc-tiotsh

larval

RNAi

All

images

are

ventral

views

with

anterior

to

the

top

a

Wild-type

adult

head

and

thorax

b

Head

and

thorax

of

Tc-tiotsh

RNAi

individual

The

basisternum

-LRB-

Bs

-RRB-

is

smaller

than

normal

c

Enlarged

view

of

region

outlined

in

A.

d

Enlarged

view

of

region

outlined

in

B

The

epimera

-LRB-

Epm

-RRB-

and

the

meso-spinasternum

-LRB-

Ss

-RRB-

are

reduced

or

missing

e

Wild-type

adult

abdomen

f

Abdomen

of

Tc-tiotsh

RNAi

adult

The

third

abdominal

segment

-LRB-

-RRB-

lacks

its

anterior

process

and

an

additional

heavily

sclerotized

segment

is

present

anterior

to

Discussion

We

have

identified

and

cloned

the

lone

member

of

the

tsh

gene

family

from

Tribolium

castaneum

Our

analysis

of

this

gene

provides

important

insights

into

the

evolution

of

the

tsh

gene

family

at

the

levels

of

both

sequence

and

function

Evolution

of

tsh

family

genes

Searches

of

several

non-drosophilid

insect

genome

sequences

identified

only

a

single

tsh

family

member

in

each

genome

These

results

corroborate

previous

suggestions

-LRB-

based

on

more

limited

data

-RRB-

that

most

insects

have

only

a

single

tsh-related

gene

and

that

this

is

the

ancestral

state

-LRB-

Herke

et

al.

Laugier

et

al.

-RRB-

A

key

implication

of

this

model

is

that

the

Drosophila

genes

tio

and

tsh

are

the

result

of

a

recent

duplication

event

that

occurred

sometime

after

the

divergence

of

mosquitoes

and

flies

Since

that

duplication

tsh

has

apparently

diverged

from

the

ancestral

sequence

faster

than

has

tio

-LRB-

although

both

have

diverged

more

than

the

Tribolium

and

Anopheles

homologs

-RRB-

More

rapid

divergence

by

one

member

of

a

recently

duplicated

gene

pair

is

not

unprecedented

For

example

the

paralogs

zerknüllt

-LRB-

zen

-RRB-

and

bicoid

-LRB-

bcd

-RRB-

are

believed

to

have

arisen

by

duplication

late

in

the

dipteran

lineage

-LRB-

Brown

et

al.

Stauber

et

al.

-RRB-

but

the

sequence

of

Zen

is

more

similar

to

that

of

the

Hox

proteins

of

other

insects

than

it

is

to

Bcd

Much

of

the

sequence

conservation

between

Tsh

family

members

is

found

in

the

zinc

finger

regions

However

a

few

additional

conserved

motifs

are

present

particularly

at

the

protein

termini

The

C-terminal

region

is

well

conserved

between

Tc-TioTsh

Ag-TioTsh

and

Tio

but

this

conserved

region

is

not

found

in

Tsh

The

conservation

of

the

N-terminal

region

is

particularly

interesting

The

Tsh

family

members

for

which

N-terminal

sequence

is

available

fall

into

two

distinct

classes

-LRB-

Fig.

-RRB-

Ag-TioTsh

and

both

Drosophila

proteins

share

an

extended

region

of

identity

that

is

completely

different

than

the

short

motif

-LRB-

MPRRKQ

-RRB-

shared

by

the

Tribolium

and

vertebrate

proteins

This

pattern

of

conservation

suggests

that

an

ancestral

Tsh

family

member

had

the

MPRRKQ

motif

which

was

replaced

with

a

different

motif

sometime

before

the

separation

of

Anopheles

and

Drosophila

within

the

dipteran

lineage

It

is

not

clear

what

the

function

of

these

disparate

motifs

might

be

as

the

vertebrate

Tsh

proteins

function

very

similarly

to

Dm-Tsh

when

expressed

in

Drosophila

-LRB-

Manfroid

et

al.

-RRB-

Perhaps

these

motifs

are

important

in

a

particular

tissue

or

at

a

different

stage

in

development

than

those

assayed

As

additional

insect

Tsh

family

protein

sequences

become

available

it

will

be

possible

to

more

accurately

determine

the

point

at

which

these

N-terminal

sequences

began

to

diverge

Evolution

of

tsh

family

gene

expression

Although

the

sequences

of

other

insect

tsh

family

members

are

most

similar

to

tio

their

expression

patterns

-LRB-

where

known

-RRB-

seem

to

be

more

similar

to

tsh

The

embryonic

expression

patterns

of

tsh

and

tio

in

Drosophila

are

strikingly

different

-LRB-

Laugier

et

al.

-RRB-

tsh

is

expressed

throughout

the

ectoderm

of

trunk

segments

while

tio

is

expressed

in

only

a

few

cells

of

the

trunk

ectoderm

-LRB-

as

well

as

in

the

Malpighian

tubules

and

parts

of

the

CNS

and

gut

-RRB-

In

tsh

mutants

however

tio

is

more

broadly

expressed

in

the

trunk

indicating

that

tsh

represses

tio

expression

in

this

region

Likewise

tsh

is

ectopically

expressed

in

the

stomodeal

region

when

tio

expression

is

knocked

down

by

RNAi

The

Tc-tiotsh

expression

pattern

includes

similiarities

to

both

the

tsh

and

tio

expression

patterns

The

ectodermal

expression

in

the

segmented

region

of

the

germband

is

reminiscent

of

although

more

extensive

than

that

of

tsh

On

the

other

hand

Tc-tiotsh

is

expressed

in

the

Malpighian

tubule

primordia

similar

to

tio

The

expression

of

tio

near

the

stomodeum

might

also

correspond

to

the

expression

of

Tc-tiotsh

in

the

labrum

and

intercalary

segment

although

it

is

difficult

to

compare

expression

patterns

in

the

cephalic

regions

of

these

insects

due

to

the

derived

structure

of

the

Drosophila

larval

head

It

is

interesting

that

the

Tc-tiotsh

expression

pattern

approximates

the

sum

of

the

tsh

and

tio

patterns

This

may

indicate

that

the

expression

pattern

of

the

ancestral

gene

that

gave

rise

to

tsh

and

tio

had

an

expression

pattern

similar

to

Tc-tiotsh

and

that

parts

of

its

expression

pattern

-LRB-

and

perhaps

its

function

-RRB-

have

been

split

between

its

daughters

Such

division

of

labor

resulting

from

complementary

loss

of

particular

expression

domains

or

functions

is

termed

subfunctionalization

and

has

been

suggested

to

play

a

key

role

in

the

maintenance

of

duplicated

genes

-LRB-

Force

et

al.

-RRB-

Among

other

insects

expression

patterns

of

tsh

family

members

have

been

reported

only

for

the

firebrat

Thermobia

domestica

and

the

milkweed

bug

Oncopeltus

fasciatus

-LRB-

Herke

et

al.

Peterson

et

al.

-RRB-

In

both

cases

these

genes

are

expressed

in

the

gnathal

thoracic

and

first

abdominal

segments

with

very

weak

-LRB-

if

any

-RRB-

expression

in

the

remainder

of

the

abdomen

Both

insects

also

show

expression

in

the

posterior

tip

of

the

embryo

-LRB-

see

below

-RRB-

Oncopeltus

has

additional

expression

in

the

procephalon

The

leg

expression

pattern

in

firebrats

has

not

been

described

but

Herke

et

al.

-LRB-

-RRB-

reported

that

milkweed

bugs

show

expression

throughout

the

legs

which

fades

from

the

distal

tips

later

in

development

Interestingly

all

of

the

insect

tsh

family

genes

whose

expression

has

been

described

are

expressed

in

the

post-abdominal

region

However

these

domains

have

been

interpreted

differently

In

firebrats

expression

in

this

region

is

reported

to

represent

the

anlagen

of

the

cerci

and

the

median

caudal

filament

-LRB-

appendages

of

the

abdominal

segment

-RRB-

-LRB-

Peterson

et

al.

-RRB-

The

posterior

expression

of

tsh

has

been

interpreted

as

the

future

anal

opening

-LRB-

Fasano

et

al.

-RRB-

and

that

of

Oncopeltus

tiptop

is

described

only

as

the

caudal

region

-LRB-

Herke

et

al.

-RRB-

Our

results

show

that

Tc-tiotsh

is

expressed

in

the

Malpighian

tubule

primordia

Likewise

tio

is

expressed

in

the

developing

Malpighian

tubules

-LRB-

Laugier

et

al.

-RRB-

Thus

Malpighian

tubule

expression

may

have

been

part

of

the

ancestral

expression

pattern

of

this

gene

family

It

is

possible

that

the

posterior

expression

domains

in

other

insects

will

upon

closer

examination

be

found

to

represent

the

developing

Malpighian

tubules

as

well

Evolution

of

tsh

family

function

Tc-tiotsh

is

required

for

the

proper

development

of

the

proximal

portion

of

developing

larval

legs

Drosophila

larvae

lack

external

appendages

but

tsh

is

expressed

in

the

proximal

regions

of

leg

imaginal

discs

and

hypomorphic

tsh

alleles

cause

proximal

leg

defects

-LRB-

Erkner

et

al.

-RRB-

These

results

suggest

that

proximal

leg

expression

and

function

were

probably

a

feature

of

a

tsh-like

gene

in

the

ancestor

of

beetles

and

flies

In

contrast

Herke

et

al.

-LRB-

-RRB-

reported

that

knockdown

of

Oncopeltus

tiptop

caused

transformations

of

the

distal

legs

to

antennae

Based

on

this

observation

they

concluded

that

Oncopeltus

tiptop

acts

as

a

distal

leg

selector

gene

and

suggested

that

tio

or

tsh

might

play

a

similar

role

in

Drosophila

tsh

expression

is

known

to

be

restricted

to

the

portion

of

the

imaginal

disc

that

will

form

the

body

wall

and

proximal

leg

segments

-LRB-

Gonzalez-Crespo

and

Morata

-RRB-

Similarly

Tc-tiotsh

is

expressed

in

the

proximal

region

of

embryonic

legs

and

leg

imaginal

discs

-LRB-

Fig.

and

data

not

shown

-RRB-

We

have

also

examined

the

expression

of

tio

in

Drosophila

leg

imaginal

discs

and

found

that

expression

is

restricted

to

the

body

wall

and

proximal

portion

of

the

developing

leg

Thus

it

is

highly

unlikely

that

the

tiptop-like

distal

leg-specifying

function

suggested

by

Herke

et

al.

-LRB-

-RRB-

is

performed

by

Tio

either

alone

or

in

combination

with

Tsh

We

can

not

rule

out

additional

internal

functions

for

Tc-TioTsh

In

particular

the

expression

of

Tc-tiotsh

in

the

Malpighian

tubules

is

intriguing

as

Drosophila

tio

-LRB-

and

possibly

other

homologs

-RRB-

show

similar

expression

The

striking

effects

of

larval

Tc-tiotsh

RNAi

would

not

have

been

predicted

based

on

Drosophila

experiments

as

relatively

few

adult

effects

of

LOF

tsh

mutations

have

been

reported

Other

than

the

proximal

leg

defects

that

have

been

discussed

above

the

only

other

adult

phenotypes

reported

are

defects

in

wing

posture

-LRB-

Soanes

et

al.

-RRB-

and

in

structures

that

develop

from

the

eye-antennal

disc

namely

the

eyes

-LRB-

Singh

et

al.

-RRB-

and

the

maxillary

palps

-LRB-

Bhojwani

et

al.

-RRB-

This

dearth

of

adult

phenotypes

could

be

a

consequence

of

the

pre-adult

lethality

of

many

tsh

allele

combinations

However

it

might

also

be

due

to

functional

redundancy

with

tio

We

examined

tio

expression

in

Drosophila

imaginal

discs

and

found

that

it

is

indeed

expressed

in

a

pattern

very

similar

to

that

of

tsh

-LRB-

data

not

shown

-RRB-

Thus

loss

of

both

tio

and

tsh

during

Drosophila

imaginal

development

might

produce

a

more

severe

phenotype

The

principal

question

prompting

our

studies

of

the

Tribolium

Tc-tiotsh

gene

was

whether

the

trunk-specifying

function

reported

for

tsh

in

Drosophila

was

conserved

in

other

insects

However

insights

from

our

work

and

other

recent

reports

compel

us

to

first

reexamine

whether

tsh

itself

is

really

a

trunk-specifying

gene

The

key

observations

which

originally

led

to

this

conclusion

were

the

presence

of

head-like

sclerites

in

embryos

lacking

tsh

and

the

positive

correlation

between

expression

of

tsh

and

the

presence

of

denticle

belts

-LRB-

a

trunk-specific

feature

-RRB-

-LRB-

de

Zulueta

et

al.

Fasano

et

al.

Roder

et

al.

-RRB-

The

later

discovery

that

tsh

and

disco

form

mutually

exclusive

domains

in

the

trunk

and

head

respectively

seemed

to

support

this

view

-LRB-

Robertson

et

al.

-RRB-

Below

we

synthesize

recent

studies

that

may

lead

to

new

interpretations

of

these

observations

A

number

of

mutant

phenotypes

in

Drosophila

including

those

of

tsh

and

some

Hox

genes

feature

small

patches

of

sclerotic

tissue

The

previous

interpretation

of

such

patches

as

ectopic

head

tissue

has

been

called

into

question

Mace

et

al.

-LRB-

-RRB-

showed

that

the

sclerites

that

form

in

a

segment

lacking

Hox

gene

expression

are

correlated

with

breaches

in

epidermal

integrity

and

the

resulting

activation

of

a

wound

response

pathway

If

in

fact

the

sclerotized

cuticle

seen

in

tsh

mutants

is

scar

tissue

rather

than

head

cuticle

the

interpretation

of

tsh

as

a

trunk-specifying

gene

would

be

less

convincing

but

still

possible

The

case

for

tsh

acting

as

a

trunk

specifier

was

apparently

strengthened

when

Robertson

et

al.

-LRB-

-RRB-

proposed

the

attractive

hypothesis

that

a

system

of

zinc

finger

proteins

acted

in

conjunction

with

the

Hox

genes

to

specify

body

regions

Within

this

system

disco

specified

gnathal

segments

and

tsh

specified

trunk

segments

However

the

same

laboratory

recently

reported

that

the

conserved

role

of

disco

is

to

specify

the

medial

portion

of

appendages

and

suggested

that

the

boundary

between

disco

and

tsh

expression

is

actually

an

artifact

of

maggot

morphology

-LRB-

Patel

et

al.

-RRB-

That

is

the

reduced

Drosophila

larval

head

might

lack

proximal

tsh-expressing

tissue

-LRB-

body

wall

-RRB-

while

retaining

medial

appendage

tissue

whereas

the

trunk

retains

tsh-expressing

tissue

but

has

lost

most

appendage

tissue

Taking

this

scenario

a

step

further

ectopic

expression

of

tsh

might

restore

the

body

wall

tissue

of

the

head

creating

larger

more

complete

segments

As

the

genes

controlling

segment

formation

also

control

denticle

belt

patterning

these

enlarged

head

segments

would

have

denticle

belts

as

well

This

interpretation

is

consistent

with

the

observation

that

the

additional

denticle

belts

produced

by

ectopic

tsh

expression

are

interspersed

with

head-specific

structures

-LRB-

de

Zulueta

et

al.

-RRB-

When

considered

in

this

light

perhaps

tsh

is

no

more

a

trunk

identity

gene

than

are

other

genes

involved

in

segment

formation

and

denticle

patterning

such

as

wingless

rhomboid

or

shavenbaby

On

the

other

hand

tsh

does

seem

to

provide

the

spatial

information

that

limits

full

segment

formation

to

the

trunk

and

thus

in

Drosophila

could

be

considered

a

trunk-specifying

gene

In

contrast

Tc-tiotsh

does

not

appear

to

act

in

any

way

as

a

trunk

identity

gene

This

is

not

to

say

that

Tc-tiotsh

has

no

role

in

the

development

of

trunk

segments

but

rather

that

we

find

no

evidence

that

it

specifies

trunk

vs

head

fates

In

embryos

depleted

of

Tc-tiotsh

the

ventral

portions

of

the

labial

and

thoracic

segments

are

reduced

but

not

visibly

transformed

Interestingly

some

of

the

labial

abnormalities

observed

in

Tc-tiotsh

RNAi

larvae

are

reminiscent

of

those

produced

by

loss

of

function

alleles

of

prothoraxless

-LRB-

Tc-Antp

-RRB-

-LRB-

Shippy

unpublished

data

-RRB-

Knockdown

of

Tc-tiotsh

during

imaginal

development

produces

adults

with

patterning

defects

in

the

thoracic

and

abdominal

sclerites

including

possible

transformations

of

anterior

abdominal

sclerites

to

a

more

posterior

identity

These

phenotypes

together

with

the

reduction

in

Tc-tiotsh

expression

seen

in

Df

-LRB-

HOMC

-RRB-

embryos

suggest

that

Tc-tiotsh

is

regulated

to

some

extent

by

Hox

genes

and

may

carry

out

a

subset

of

the

functions

of

these

genes

in

both

the

head

and

the

trunk

Our

studies

of

Tc-tiotsh

indicate

that

trunk

specification

is

not

a

universal

function

of

tsh

family

members

It

is

possible

that

this

function

has

been

lost

in

Tribolium

However

expression

pattern

data

from

other

insects

and

functional

analysis

in

Oncopeltus

argues

against

this

conclusion

A

more

likely

scenario

is

that

tsh

family

members

acquired

a

role

in

defining

trunk

at

some

point

after

the

divergence

of

the

coleopteran

and

dipteran

lineages

Analysis

of

tsh

function

in

additional

holometabolous

insects

should

help

pinpoint

when

this

event

occurred

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

Fig

Dynamic

expression

of

Tc-tiotsh

during

germband

elongation

Tc-tiotsh

expression

always

appears

in

an

odd-numbered

parasegment

and

the

anterior

compartment

of

the

adjacent

posterior

even-numbered

parasegment

before

it

is

detected

in

the

posterior

compartment

of

the

adjacent

anterior

even-numbered

parasegment

An

arrowhead

marks

the

mandibular

Engrailed

stripe

in

each

embryo

while

numbers

indicate

parasegment

identity

aTc-tiotsh

expression

is

first

observed

spanning

the

mandibular

Engrailed

stripe

The

mesoderm

which

has

not

yet

invaginated

at

the

ventral

midline

does

not

show

Tc-tiotsh

expression

bTc-tiotsh

is

expressed

in

and

the

anterior

compartment

of

but

not

yet

in

the

posterior

compartment

of

c

An

embryo

with

a

gap

in

Tc-tiotsh

expression

in

the

posterior

compartment

of

Tc-tiotsh

expression

is

also

visible

in

the

intercalary

segment

and

the

developing

labrum

d

An

embryo

in

which

the

gap

in

Tc-tiotsh

expression

corresponds

to

the

posterior

compartment

of

-LRB-

GIF

kb

-RRB-

Fig

Tc-tiotsh

and

Tc-Even-skipped

-LRB-

Tc-Eve

-RRB-

expression

Tc-Eve

expression

is

shown

in

brown

while

Tc-tiotsh

expression

is

in

purple

Embryos

are

shown

in

ventral

view

with

anterior

to

the

left

Numbers

refer

to

Tc-Eve

stripes

a

The

first

Tc-Eve

primary

stripe

has

resolved

into

two

secondary

stripes

and

The

Tc-tiotsh

expression

domain

at

this

time

covers

not

only

the

Tc-Eve

first

primary

stripe

region

but

also

the

region

anterior

to

it

The

second

Tc-Eve

primary

stripe

has

formed

too

but

no

Tc-tiotsh

expression

is

present

in

this

region

yet

Tc-tiotsh

is

also

expressed

in

the

amnion

b

The

second

Tc-Eve

primary

stripe

is

resolving

into

and

but

there

is

still

no

Tc-tiotsh

expression

in

this

region

The

expression

of

Tc-tiotsh

in

the

amnion

is

still

visible

at

this

stage

c

The

third

Tc-Eve

primary

stripe

has

formed

and

is

just

beginning

to

split

into

secondary

stripes

A

new

stripe

of

Tc-tiotsh

expression

covers

the

region

delimited

by

Tc-Eve

stripes

and

-LRB-

and

the

anterior

compartment

of

-RRB-

No

Tc-tiotsh

expression

is

seen

in

the

posterior

compartment

of

yet

d

A

much

older

embryo

than

in

A

B

and

C

shows

the

same

relationship

between

Tc-Eve

and

Tc-tiotsh

expression

in

the

posterior

region

of

the

embryo

-LRB-

GIF

kb

-RRB-

Fig

Tc-sal

expression

Anterior

is

to

the

top

In

a

wild-type

embryo

in

which

the

abdominal

segments

are

just

beginning

to

form

Tc-sal

is

expressed

in

most

of

the

gnathal

and

thoracic

segments

-LRB-

GIF

kb

-RRB-

O B O O O B O O O O O O O O O B O O O B 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 B 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 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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 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 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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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tribolium teashirt tiptop trunk homeotic


Secondary meningioma in a long-term survivor of atypical teratoid/rhabdoid tumour with a germline INI1 mutation Objective We report on a patient who developed a meningioma more than two decades after removal at a young age of an atypical teratoid/rhabdoid tumour -LRB- AT/RT -RRB- , which was due to a germline INI1 mutation , and radio - and chemotherapy . Introduction We previously reported on a family in which four cousins developed an atypical teratoid/rhabdoid tumour -LRB- AT/RT -RRB- at young age , due to inheritance of a germline G > A mutation in the donor splice site of exon 4 of the tumour suppressor gene INI1 -LSB- 2 -RSB- . More than 20 years after resection of the AT/RT , the oldest of the four cousins developed an intracranial meningioma and , after an additional year , a myoepithelioma of the lip . In addition to the constitutional INI1 mutation and loss of the normal INI1 allele , we detected an identical rearrangement in the NF2 gene , not affecting its coding regions , in the AT/RT and myoepithelioma of the patient and concluded that they both originated from a common precursor cell . However , the rearrangement in NF2 and loss of the normal INI1 allele could not be demonstrated in the meningioma . Despite this , the development of the meningioma may be the consequence of the presence of the constitutional INI1 mutation , because mutations in this gene have been identified in meningiomas -LSB- 8 -RSB- . Alternatively , the meningioma could be induced by the radiation therapy , which our patient received after removal of the AT/RT and which is a well-known consequence of this treatment -LSB- 1 -RSB- . We investigated the meningioma for the presence of genetic changes reported in radiation-induced meningiomas and performed a comparative loss of heterozygosity -LRB- LOH -RRB- analysis to demonstrate that such radiation-induced genetic changes are absent in the AT/RT and myoepithelioma of our patient . Materials and methods Tumour samples and DNA isolation DNA from the AT/RT and the myoepithelioma was isolated from formalin-fixed and paraffin-embedded tumour material , and DNA from the meningioma was isolated from a fresh frozen tumour sample using commercially available kits -LRB- Qiagen , Venlo , The Netherlands -RRB- . SNP analysis A genome-wide single nucleotide polymorphism -LRB- SNP -RRB- analysis of normal and meningioma DNA was conducted with the Affymetrix GeneChip Human Mapping 10K Array Xba 142 2.0 . The array contains 10,204 SNPs distributed across the genome . The GeneChips were processed by an authorised Affymetrix Service Provider using standard Affymetrix protocols -LRB- Service XS , Leiden , The Netherlands -RRB- . LOH regions were identified by comparing the SNP profiles of normal and meningioma DNA and scoring heterozygous SNP markers -LRB- AB -RRB- in the normal DNA that were reduced to homozygosity -LRB- AA or BB -RRB- in the meningioma DNA . Intervals were determined on the basis of locations of SNP markers in the UCSC Genome Browser , March 2006 Build , at http://genome.ucsc.edu/ . LOH analysis Loss of heterozygosity -LRB- LOH -RRB- analysis was performed with microsatellite markers as described previously -LSB- 3 -RSB- using primer sequences and conditions for polymerase chain reaction given by The Genome Database at http://www.gdb.org/ . Because of rather extensive degradation of DNA extracted from the AT/RT , only markers generating PCR fragments smaller than 150 bp were used . Case report History Our patient was diagnosed with a tumour in the fourth ventricle and right cerebellar hemisphere at the age of 4.5 years . Resection of the tumour was incomplete . The tumour was originally classified as anaplastic ependymoma WHO grade III , but upon extensive genetic analysis and histopathological re-evaluation , reclassified as AT/RT -LSB- 2 -RSB- . He received adjuvant chemotherapy -LRB- methotrexate , vincristine and prednisolone -RRB- followed by craniospinal radiotherapy -LRB- 3,300 cGy in 22 doses -RRB- with a boost of 2,100 cGy on the location of the tumour . Fifteen years later , a brain CT without contrast showed no signs of recurrent disease or of other new pathology . Almost 6 years later , 20.5 years after initial diagnosis , he presented again with bifrontal headaches , increasing ataxic gait disturbances and fatigue . CT examination showed a large isodens space-occupying lesion in the left temporal region with oedema , midline shift and contrast enhancement . One year after resection of this tumour , he developed a myoepithelioma of the upper lip . The patient is alive at 29 years of age . Histological findings Histopathological examination of the left temporal tumour revealed an atypical meningothelial meningioma WHO grade II with many vessels and up to six mitoses per 2 mm2 . SNP and LOH analysis Radiation-induced meningiomas usually display a complex karyotype -LSB- 1 -RSB- . To determine the genetic changes , we performed a genome-wide scan by SNP analysis of the meningioma and corresponding normal DNA . The identified chromosomal regions displaying LOH are listed in Table 1 . Next , we tested several markers from within each of the five largest deleted regions by conventional microsatellite analysis to determine their LOH status in the meningioma and in the AT/RT and myoepithelioma . Presence of LOH in the meningioma , but absence of LOH in AT/RT and myoepithelioma was found for D1S199 in 1pter-p34.3 -LRB- Fig. 1a -RRB- , D2S327 in 2pter-p12 , D3S3727 in 3p26.1-cen -LRB- Fig. 1b -RRB- , D16S3020 in 16pter-p12.1 and D17S1872 in 17qcen-q21.31 -LRB- Fig. 1c -RRB- . In contrast , as determined previously -LSB- 2 -RSB- and exemplified by marker D22S430 in Fig. 1d , there was LOH for chromosome 22 markers in the AT/RT and myoepithelioma , but not in the meningioma . Fig. 1LOH analysis using markers from chromosome regions a 1pter-p34.3 -LRB- D1S199 -RRB- , b 3p26.1-cen -LRB- D3S3727 -RRB- , c 17qcen-q21.31 -LRB- D17S1872 -RRB- and d 22q -LRB- D22S430 -RRB- of AT/RT -LRB- A -RRB- , meningioma -LRB- M -RRB- and myoepithelioma -LRB- My -RRB- DNAs of the patient . N denotes normal DNA derived from the patient 's blood leucocytes . Arrowhead indicates position of lost alleleTable 1LOH regions in the meningioma , as determined by SNP analysisChromosome regionExact interval -LRB- Mb -RRB- 1pter-p34.30 -- 38.901p33-p3346.99 -- 51.091p32.2-1p31.358 .19 -- 65.502pter-p120 -- 79.283p26.1-cen6.11 -- 88.847q36.1-qter150.04 -- 158.6616pter-p12.10 -- 22.7516q23.1-q23.373 .46 -- 81.3317cen-q21.3123 .09 -- 40.3519pter-p13.20 -- 8.83 Discussion In a previous study , we provided evidence for the involvement of INI1 , by constitutional mutation and independent somatic loss , in the development of both the AT/RT and the myoepithelioma of our patient . Moreover , we could demonstrate that these tumours originated from an identical precursor cell -LSB- 2 -RSB- . Although involvement of INI1 has been described in sporadic meningiomas -LSB- 8 -RSB- , until now meningiomas have not been reported to occur as a consequence of the inheritance of an INI1 mutation . However , this might be due to the extreme rarity of families with an inherited INI1 mutation , with only four published cases so far -LSB- 2 , 5 , 6 , 11 -RSB- . In this context , it is interesting to note that -LRB- as demonstrated before and exemplified by marker D22S430 in Fig. 1d -RRB- the meningioma of our patient has no LOH 22 and the retained INI1 allele carries no additional inactivating mutation -LSB- 2 -RSB- , indicating that this tumour is not the result of inheritance of the mutated INI1 gene . In contrast , the meningioma displayed genetic changes that are characteristic for radiation-induced meningiomas -LSB- 1 , 7 , 9 -RSB- , such as a complex karyotype , as evidenced by the SNP analysis -LRB- Table 1 -RRB- , including extensive losses on chromosome arm 1p , but no loss of chromosome 22 , and no mutation in the NF2 gene -LSB- 2 -RSB- . The genetic changes found in the meningioma proved not to be present in the AT/RT or myoepithelioma -LRB- Fig. 1a -- c , and data not shown -RRB- , demonstrating that the meningioma is genetically not related to these tumours and should not be considered as a late and more differentiated recurrence or metastasis of the AT/RT . In conclusion , the clinical and genetic characteristics of the meningioma clearly indicate that the tumour developed as a consequence of the radiation therapy . Recent evidence suggests that long-term survival of patients with a childhood AT/RT can occur especially after aggressive treatment including radiotherapy -LSB- 10 , 12 -RSB- . The possible late effects of radiation therapy have been considered as serious drawbacks of this treatment . This is the first case in literature in which such a possible consequence in the form of a radiation-induced meningioma is actually documented in a long-term survivor of an AT/RT . Compared to sporadic meningiomas , radiation-induced meningiomas are more aggressive and have a higher chance to recur -LSB- 1 -RSB- . Because of this unfavourable prognosis , continuous medical surveillance of our patient is warranted .

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atypical teratoid/rhabdoid tumour
genetic predisposition