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induction of ectopic eyes by targeted expression, Papers of Developmental biology

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bg1
mun.
163,
378
(1989).
81.
E.
Munozetal.,
Eur.
J.
Immunol.
22,1391
(1993);
M.
Kohmo
et
al.,
Biochem.
J.
267,
91
(1990);
A.
V.
Hoffbard
and
R.
G.
Wickremusingine,
Blood
75,
88
(1990);
G.
R.
Guy
et
al.,
J.
Biol.
Chem.
266,
14343
(1991).
82.
Z.
Dong
et
al.,
J.
Immunol.
151,
2717
(1993);
T.
Iwasaki
et
al.,
FEBS
Lett.
298,
240
(1992).
83.
R.
N.
Maini
etal.,
Clin.
Exp.
Rheumatol.
11
(suppl.
8),
S173
(1993);
F.
M.
Brennan,
R.
N.
Maini,
M.
Feld-
mann,
J.
Rheumatol.
31,
293
(1992).
84.
C.
Tsai
et
al.,
in
preparation.
85.
M.
Dukes
et
al.,
J.
Endocrinol.
135,
239
(1992).
86.
S.
R.
Hubbard
et
al.,
Nature
372,
746
(1994).
87.
A.
Levitzki,
Eur.
J.
Biochem.
226,
1
(1994).
88.
The
studies
from
the
author's
laboratory
were
sup-
ported
by
The
Konover
Cancer
Fund
of
Hebrew
Uni-
versity,
by
the
Ministry
of
Health
of
Israel,
and
by
SUGEN,
Redwood
City,
CA.
The
authors
also
thank
Martha
Velorde
of
SUGEN
for
assistance
in
prepar-
ing
the
manuscript.
RESEARCH
ARTICLE
Induction
of
Ectopic
Eyes
by
Targeted
Expression
of
the
eyeless
Gene
in
Drosophila
Georg
Halder,*
Patrick
Callaerts,*
Walter
J.
Gehringt
The
Drosophila
gene
eyeless
(ey)
encodes
a
transcription
factor
with
both
a
paired
domain
and
a
homeodomain.
It
is
homologous
to
the
mouse
Small
eye
(Pax-6)
gene
and
to
the
Aniridia
gene
in
humans.
These
genes
share
extensive
sequence
identity,
the
position
of
three
intron
splice
sites
is
conserved,
and
these
genes
are
expressed
similarly
in
the
developing
nervous
system
and
in
the
eye
during
morphogenesis.
Loss-of-function
mu-
tations
in
both
the
insect
and
in
the
mammalian
genes
have
been
shown
to
lead
to
a
reduction
or
absence
of
eye
structures,
which
suggests
that
ey
functions
in
eye
mor-
phogenesis.
By
targeted
expression
of
the
ey
complementary
DNA
in
various
imaginal
disc
primordia
of
Drosophila,
ectopic
eye
structures
were
induced
on
the
wings,
the
legs,
and
on
the
antennae.
The
ectopic
eyes
appeared
morphologically
normal
and
consisted
of
groups
of
fully
differentiated
ommatidia
with
a
complete
set
of
photoreceptor
cells.
These
results
support
the
proposition
that
ey
is
the
master
control
gene
for
eye
morphogenesis.
Because
homologous
genes
are
present
in
vertebrates,
ascidians,
insects,
cephalopods,
and
nemerteans,
ey
may
function
as
a
master
control
gene
throughout
the
metazoa.
The
eyeless
(ey)
mutation
of
Drosophila
was
first
described
in
1915
(1)
on
the
basis
of
its
characteristic
phenotype,
the
partial
or
com-
plete
absence
of
the
compound
eyes.
The
ey
alleles
available
today
are
recessive
hypo-
morphs
(weak
alleles)
and
they
lead
to
the
reduction
or
complete
absence
of
the
com-
pound
eyes
but
do
not
affect
the
ocelli
(sim-
ple
eyes)
on
the
head
of
the
fly.
Apparent
null
alleles
that
are
lethal
when
homozygous
have
also
been
isolated
(2),
but
they
have
been
lost,
and
a
detailed
analysis
of
their
phenotype
is
not
available.
Cloning
and
se-
quencing
of
the
ey
gene
(3)
have
shown
that
it
encodes
a
transcription
factor
that
con-
tains
both
a
paired
domain
and
a
homeodo-
main.
The
ey
gene
is
homologous
to
Small
eye
(Sey
=
Pax-6)
in
the
mouse
and
to
Aniridia
in
humans.
The
proteins
encoded
by
these
genes
share
94
percent
sequence
iden-
tity
in
the
paired
domain,
and
90
percent
identity
in
the
homeodomain
and
they
con-
tain
additional
similarities
in
the
flanking
sequences.
Furthermore,
two
out
of
three
splice
sites
in
the
paired
box
and
one
out
of
two
splice
sites
in
the
homeobox
are
con-
served
between
the
Drosophila
and
the
mam-
malian
genes,
which
indicates
that
these
genes
are
orthologous.
Both
the
mouse
and
the
Drosophila
gene
have
similar
expression
patterns
during
de-
velopment.
In
the
mouse,
the
expression
of
Sey
is
observed
in
the
spinal
cord,
in
discrete
regions
of
the
brain,
and
in
the
developing
eye.
The
Sey
gene
is
expressed
from
the
earliest
stages
until
the
end
of
eye
morpho-
genesis:
first,
in
the
optic
sulcus,
and
subse-
quently
in
the
eye
vesicle,
in
the
lens,
in
the
differentiating
retina,
and
finally
in
the
cor-
nea
(4).
In
Drosophila,
ey
is
first
expressed
in
the
embryonic
ventral
nerve
cord
and
in
defined
regions
of
the
brain.
Later
in
embry-
ogenesis,
ey
is
transcribed
in
the
embryonic
primordia
of
the
eye
as
soon
as
these
cells
can
be
detected.
In
subsequent
larval
stages,
it
continues
to
be
expressed
in
the
develop-
ing
eye
imaginal
discs.
During
the
third
lar-
val
stage,
ey
expression
becomes
largely
re-
stricted
to
the
part
of
the
eye
disc
that
is
anterior
to
the
morphogenetic
furrow.
This
region
consists
of
undifferentiated
cells
whereas
posterior
to
the
furrow
the
differen-
tiating
ommatidia
are
apparent
(5).
Because
mutations
in
the
mouse
and
Drosophila
genes
lead
to
a
reduction
or
complete
absence
of
all
eye
structures,
and
because
these
genes
are
similar
in
DNA
sequence
and
in
expression
pattern
even
at
the
earliest
stage
of
eye
de-
velopment,
it
has
been
suggested
that
ey
and
Sey
may
be
the
master
control
genes
in-
volved
in
eye
morphogenesis
(3).
Further-
more,
mutations
in
four
other
Drosophila
genes
with
similar
phenotypes
(eyes
absent,
sine
oculis,
eye
gone,
and
eyelisch)
do
not
affect
the
expression
pattern
of
ey,
which
indicates
that
ey
acts
upstream
of
these
other
genes
(6).
These
results
are
consistent
with
its
possible
role
as
a
gene
that
controls
eye
morphogenesis,
even
though
it
may
have
additional
functions
in
the
developing
ner-
vous
system.
The
cloning
of
the
homologous
genes
from
ascidians,
cephalopods,
and
nem-
erteans
(ribbon
worms)
suggests
that
this
gene
may
be
present
in
all
metazoa
(3).
Master
control
genes
that
act
as
develop-
mental
switches
can
be
detected
on
the
basis
of
their
mutant
phenotypes.
Thus,
homeotic
mutations
have
identified
master
control
genes
that
specify
the
body
plan
along
the
antero-posterior
axis.
These
genes,
which
are
characterized
by
a
homeobox,
are
clustered
in
the
Antennapedia
(Antp)
and
Bithorax
Complexes
in
Drosophila,
and
in
the
Hox
gene
clusters
of
the
mouse
(7).
Loss-
and
gain-of-function
mutations
in
these
genes
lead
to
opposite
homeotic
transformations.
For
example,
in
Antp,
recessive
loss-of-func-
tion
mutations
are
lethal
at
the
embryonic
or
larval
stage
and
lead
to
a
transformation
of
the
second
thoracic
segment
(T2)
toward
the
first
thoracic
segment
(T2->T1).
Dom-
inant
gain-of-function
mutations
lead
to
a
transformation
in
the
opposite
direction,
that
is
from
the
anterior
head
and
T1
seg-
ments
toward
T2
(H,T1->T2)
(8).
These
transformations
can
be
explained
by
the
combinatorial
interaction
of
several
ho-
meotic
genes
in
order
to
specify
a
given
body
segment.
These
genes
have
partially
overlap-
ping
expression
domains
in
several
body
seg-
ments
and
each
segment
is
specified
by
a
combination
of
homeobox
genes,
that
is
by
a
Hox
code
(9).
By
ubiquitous
(ectopic)
ex-
pression
of
Antp
under
the
control
of
a
heat-
shock
promoter,
we
have
changed
the
body
plan
of
Drosophila
and
induced
the
formation
of
middle
legs
in
place
of
the
antennae,
and
SCIENCE
*
VOL.
267
·
24
MARCH
1995
Biozentrum,
University
of
Basel,
Klingelbergstrasse
70,
CH-4056
Basel,
Switzerland.
*The
first
two
authors
contributed
equally
to
this
work.
tTo
whom
correspondence
should
be
addressed.
IPPLYIIUrlllll.81131BBl.li.BiB.
1788
Downloaded from https://www.science.org at University of Sussex on April 13, 2023
pf3
pf4
pf5

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Download induction of ectopic eyes by targeted expression and more Papers Developmental biology in PDF only on Docsity!

mun. (^) 163, 378 (1989).

  1. E. (^) Munozetal., Eur. J. Immunol. (^) 22,1391 (^) (1993); M. Kohmo et (^) al., Biochem. J. 267, (^91) (1990); A. V. Hoffbard and R. G. (^) Wickremusingine, Blood (^) 75, 88 (1990); G.^ R.^ Guy et^ al.,^ J. Biol.^ Chem.^ 266,^14343 (1991).
  2. Z. (^) Dong et al., J. Immunol. 151, (^2717) (1993); T.

Iwasaki et (^) al., FEBS Lett. (^) 298, 240 (1992).

  1. R. N. Maini (^) etal., Clin. (^) Exp. Rheumatol. (^11) (suppl. 8), S173 (^) (1993); F. M.^ Brennan, R. N.^ Maini, M. Feld- mann, J. Rheumatol. (^) 31, 293 (1992).
  2. C. Tsai et (^) al., in (^) preparation.
  3. M. Dukes et al., J. Endocrinol. 135, (^239) (1992).
  4. S. R. Hubbard et al., Nature 372, (^746) (1994).
    1. A. (^) Levitzki, Eur. J. Biochem. (^) 226, 1 (1994).
    2. The studies from the author's (^) laboratory were (^) sup- ported by The^ Konover^ Cancer^ Fund^ of^ Hebrew^ Uni- versity, by the^ Ministry of Health of^ Israel, and^ by SUGEN, Redwood^ City, CA.^ The^ authors also^ thank Martha Velorde of SUGEN for assistance in (^) prepar- ing the^ manuscript.

RESEARCH ARTICLE

Induction of (^) Ectopic Eyes by

Targeted Expression of

the (^) eyeless Gene in (^) Drosophila

Georg Halder,^ Patrick^ Callaerts,^ Walter^ J.^ Gehringt

The Drosophila gene eyeless (ey) encodes a transcription factor with both a paired domain

and a homeodomain. It is homologous to the mouse Small eye (Pax-6) gene and to the

Aniridia (^) gene in humans. These (^) genes share extensive (^) sequence identity, the (^) position of three intron (^) splice sites is (^) conserved, and these (^) genes are (^) expressed (^) similarly in the developing nervous^ system and^ in^ the^ eye during morphogenesis. Loss-of-function mu- tations in both the insect and in the mammalian (^) genes have been shown to lead to a reduction or absence of (^) eye structures, which (^) suggests that (^) ey functions in (^) eye mor- phogenesis. By targeted expression of the^ ey complementary DNA in various^ imaginal disc primordia of^ Drosophila, ectopic eye structures^ were induced on the^ wings, the^ legs, and

on the antennae. The ectopic eyes appeared morphologically normal and consisted of

groups of^ fully differentiated ommatidia^ with^ a^ complete set of^ photoreceptor cells. These

results support the proposition that ey is the master control gene for eye morphogenesis.

Because (^) homologous genes are (^) present in (^) vertebrates, ascidians, insects, (^) cephalopods, and (^) nemerteans, ey may function as a master control (^) gene throughout the metazoa.

The (^) eyeless (ey) mutation of (^) Drosophila was

first described in 1915 (1) on the basis of its

characteristic (^) phenotype, the (^) partial or com- plete absence^ of^ the^ compound eyes. The^ ey alleles available (^) today are recessive (^) hypo- morphs (weak^ alleles) and^ they lead^ to^ the reduction or (^) complete absence of the com- pound eyes but do^ not^ affect the ocelli^ (sim- ple eyes) on^ the head^ of^ the^ fly. Apparent null alleles that are lethal when (^) homozygous have also been isolated (^) (2), but (^) they have been (^) lost, and a detailed (^) analysis of their phenotype is^ not^ available.^ Cloning and^ se-

quencing of^ the^ ey gene (3) have shown that

it encodes a transcription factor that con-

tains both a (^) paired domain and a homeodo- main. The (^) ey gene is (^) homologous to Small

eye (Sey =^ Pax-6) in^ the^ mouse^ and^ to Aniridia in humans. The (^) proteins encoded (^) by

these genes share 94 percent sequence iden-

tity in^ the^ paired domain, and^90 percent identity in^ the homeodomain and^ they con-

tain additional similarities^ in^ the^ flanking

sequences. Furthermore,^ two^ out^ of^ three

splice sites^ in^ the^ paired box and^ one^ out^ of two (^) splice sites in the homeobox are con- served between the (^) Drosophila and the mam- malian (^) genes, which indicates^ that these genes are^ orthologous. Both the mouse and the (^) Drosophila gene have similar (^) expression patterns during de- velopment. In^ the^ mouse, the^ expression of Sey is^ observed^ in^ the^ spinal cord,^ in^ discrete regions of the^ brain, and^ in^ the^ developing eye. The^ Sey gene is^ expressed from the earliest (^) stages until the end of (^) eye morpho- genesis: first,^ in^ the^ optic sulcus,^ and^ subse- quently in^ the^ eye vesicle, in^ the^ lens, in^ the differentiating retina,^ and^ finally in^ the^ cor- nea (^) (4). In (^) Drosophila, ey is first (^) expressed in the (^) embryonic ventral nerve cord and in defined (^) regions of the brain. Later in (^) embry- ogenesis, ey is^ transcribed^ in^ the^ embryonic primordia of the^ eye as soon as^ these cells can be detected. In (^) subsequent larval (^) stages, it continues to be (^) expressed in the (^) develop- ing eye imaginal discs.^ During the third lar- val (^) stage, ey expression becomes (^) largely re- stricted to the (^) part of the (^) eye disc that is

anterior to the (^) morphogenetic furrow. This

region consists^ of^ undifferentiated^ cells

whereas posterior to the furrow the differen-

tiating ommatidia^ are^ apparent (5).^ Because

mutations in the mouse and (^) Drosophila genes lead to a reduction or (^) complete absence of all

eye structures,^ and because these^ genes are

similar in DNA sequence and in expression

pattern even^ at^ the earliest^ stage of^ eye de-

velopment, it^ has been^ suggested that^ ey and Sey may be the^ master^ control^ genes in- volved in eye morphogenesis (3). Further- more, mutations^ in^ four^ other^ Drosophila genes with^ similar^ phenotypes (eyes absent, sine (^) oculis, (^) eye gone, and (^) eyelisch) do not

affect the expression pattern of ey, which

indicates that (^) ey acts (^) upstream of these other

genes (6).^ These^ results^ are^ consistent^ with

its (^) possible role (^) as a (^) gene that controls (^) eye morphogenesis, even^ though it may have additional functions in the (^) developing ner- vous (^) system. The (^) cloning of the (^) homologous genes from^ ascidians,^ cephalopods, and^ nem-

erteans (ribbon worms) suggests that this

gene may be^ present in^ all^ metazoa^ (3).

Master control (^) genes that act as (^) develop- mental switches can be detected on the basis of their mutant (^) phenotypes. Thus, homeotic mutations have identified master control genes that^ specify the^ body plan along the

antero-posterior axis.^ These^ genes, which^ are

characterized (^) by a (^) homeobox, are clustered in the (^) Antennapedia (Antp) and Bithorax Complexes in^ Drosophila, and^ in^ the^ Hox

gene clusters^ of^ the^ mouse^ (7). Loss-^ and

gain-of-function mutations in^ these^ genes

lead to opposite homeotic transformations.

For (^) example, in (^) Antp, recessive loss-of-func- tion mutations are lethal at the (^) embryonic or larval (^) stage and lead to a transformation of

the second thoracic segment (T2) toward

the first thoracic segment (T2->T1). Dom-

inant (^) gain-of-function mutations lead to a

transformation in the opposite direction,

that is from the (^) anterior head and (^) T1 seg- ments toward T2 (^) (H,T1->T2) (^) (8). These transformations can be (^) explained by the combinatorial interaction of several ho- meotic genes in order to (^) specify a (^) given body segment. These^ genes have^ partially overlap- ping expression domains^ in^ several^ body seg- ments and each (^) segment is (^) specified by a combination of homeobox (^) genes, that is (^) by a Hox code (^) (9). (^) By ubiquitous (ectopic) ex-

pression of^ Antp under the control^ of^ a^ heat-

shock (^) promoter, we^ have^ changed the^ body plan of^ Drosophila and induced the^ formation

of middle legs in place of the antennae, and

SCIENCE *^ VOL.^267 · 24 MARCH 1995

Biozentrum, (^) University of Basel, (^) Klingelbergstrasse 70, CH-4056 Basel, Switzerland. *The first two authors contributed (^) equally to this work. tTo whom^ correspondence should be^ addressed.

IPPLYIIUrlllll.81131BBl.li.BiB.

1788

Downloaded from https://www.science.org at University of Sussex on April 13, 2023

genes indicate that^ there^ is^ competition be- tween (^) the (^) ectopically expressed gene and the genes normally expressed in^ a^ given segment (11). This^ competition frequently leads to

epistasis of the^ posterior over^ the^ anterior

genes, and^ to^ segmental transformations^ that are confined to the anterior (^) body segments.

GAL 4

t~~~~~~~~~~~~~~~~~~~~~~~~~

--00000-I eye/ess^ embryonic^ cDNA-

UAS Transcription of eyeless in antennal, leg and^ wing imaginal discs

Fig. 1.^ Targeted expression of ey. (A) Schematic^ representation of^ the^ ectopic induction^ of^ ey by means of
the GAL4 system. In (B) through (D), f3-galactosidase staining of third instar imaginal discs (28) shows the

activation of a (^) UAS-lacZ reporter construct (^) by the GAL4 (^) enhancer-trap line (^) E132. (^) (B) Eye-antennal disc.

The antennal portion of the disc is on the top and the eye portion is on the bottom. 13-Galactosidase activity
is detected in parts of the antennal disc corresponding to several antennal segments and in the periphery
of the disc, which will give rise to head cuticle. The staining observed at the most posterior part of the eye
disc derives from the optic nerve. (C) Wing imaginal disc. 13-Galactosidase activity is detected in proximal
regions of^ the^ future^ wing blade, and in^ portions corresponding to the hinge regions and ventral pleura. (D)
Leg imaginal disc with^ lacZ expression in^ portions that^ correspond to the tibia and femur.

Fig. 2. GAL4 driven ectopic expression of ey in- duces the formation of (^) eye structures in (^) variousm tissues. The sites at which ectopic eyes form cor- respond to the regions in the imaginal discs, in which GAL4 is^ expressed as assayed by the acti-

vation of a lacZ reporter construct (Fig. 1, B, C,

and D). The ectopic eye structures show omma- tidial arrays, (^) interommatidial bristles, and red pig-

mentation (29).^ (A) Cuticle^ of an adult^ head in

which both antennae formed eye structures. (B)

Dissected wing (^) with a large outgrowth of eye tis- sue. The ectopic eye contains about 350 (^) facets.

Many interommatidial bristles are also apparent.

The normal eye contains approximately (^800) om- matidia. The wing is reduced in size. The anterior margin with its characteristic triple row of bristles occupies most of the circumference, whereas the more posterior (^) structures are absent and re- placed (^) by eye tissue. The characteristic venation pattern of the wing is disturbed by the formation (^) of the ectopic eye structures. (C) Dissected antenna in which most of the third antennal segment is replaced by eye structures. (D) Dissected middle leg with an eye-outgrowth on the base of the (^) tibia.

The ey gene, which also contains (^) a ho- meobox in addition to a (^) paired box, differs from Antp and the other (^) antero-posterior homeotic genes in that the (^) hypomorphic

loss-of-function mutation leads to a loss of

the corresponding (^) eye structures rather (^) than to their homeotic transformation. This phe- notype does not (^) necessarily imply that (^) ey acts as a developmental switch; it only shows that ey function is (^) required for eye develop- ment. If, however, ey is the master control gene (^) for eye morphogenesis, the ectopic ex-

pression of ey should induce the formation of

ectopic eye structures in other (^) parts of the body similar^ to the transformations^ obtained for (^) Antp (10) and the other homeotic (^) genes (11). Therefore we used the GAL4 (^) system (12) and a heat-inducible expression vector in order to (^) express the (^) ey gene ectopically. Induction of ectopic eye structures. We used the GAL4 (^) system (12) to (^) target ey expression to various (^) imaginal discs (^) other than the (^) eye discs (^) in which ey is (^) normally expressed. GAL4 is a (^) yeast transcriptional

activator that can activate transcription of

any gene after^ introduction^ into^ Drosophila if the (^) gene is (^) preceded by a GAL4 (^) upstream activating sequence (UAS) that consists of five (^) optimized GAL4 binding sites (12). The GAL4 system is now (^) widely used in (^) conjunc-

tion with a method called enhancer detec-

tion (13), in which a (^) reporter gene is (^) pro- vided with a weak promoter (^) only and insert- ed at random sites (^) in the genome by trans-

position. If the detector has inserted close to

an enhancer, the (^) reporter gene is (^) expressed differentially. (^) By isolating a (^) large number (^) of

enhancer detection lines, a spectrum of dif-

ferent enhancers with (^) specific temporal and

SCIENCE · VOL. 267 · 24 MARCH 1995

also transformed the dorsal head (^) capsule into structures of the second thoracic (^) segment (H->T2). This^ phenotype is^ similar to that observed in dominant (^) gain-of-function muta- tions (^) (10). However, it (^) proved to be difficult to transform the more (^) posterior body seg-

ments toward T2. Data for several homeotic

iAL

  • Genomic^ enhancer-VGL4j-

Tissue-specific expression of GAL 4

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S AS

ASa a

Fig. 4.^ Histological structure^ and^ differentiation of^ photoreceptors in^ the^ ectopic eye. (A) Micrograph of a
section through an ectopic eye in the antenna (to the left) and the normal eye (to the right) stained with Azur
II and methylene blue (15). (B) Phase contrast micrograph of a section through an ectopic eye on the
antenna. The normal number^ and^ trapezoidal arrangement of the rhabdomeres of^ photoreceptors is
observed in the different ommatidia (arrowhead). (C) Micrograph of an eye-antennal disc stained with an
antibody against the neuronal marker^ ELAV^ and a^ secondary fluorescein-labeled^ antibody. In^ the normal

eye portion (to the^ right), regularly spaced^ ommatidial clusters^ of^ differentiating^ photoreceptors^ are detect-

ed. In the antennal part of the disc (on the left), extensive cell proliferation has led to a^ doubling in^ size.^ In this

portion, a^ large domain^ of^ ectopically induced^ photoreceptors is seen.^ (D)^ and^ (E)^ are^ higher magnification

views of (C), which shows the photoreceptor clusters in the ectopic eye (D) and in^ the normal eye (E),

respectively. An^ essentially normal cluster formation^ and cluster^ array is observed^ in the^ ectopic eye.

were seen at one side^ of an^ ectopic photore- ceptor cluster. This^ expression most^ likely corresponds to the formation^ of^ Rs^ photore- ceptor cells.^ Subsequently, groups of^ three, five, seven, and^ eight cells were detected that (^) expressed the ELAV (^) epitope. This series of events (^) probably corresponds to what is observed in a normal (^) eye disc (^) upon passage of the (^) morphogenetic furrow. (^) Thus, these observations (^) suggest that^ morphogenesis of the (^) ectopic eyes is normal and that it (^) prob- ably involves the formation^ of an^ ectopic morphogenetic furrow.^ In^ summary, the data presented above show^ that^ ey^ can induce the formation of (^) complete and (^) morphologically normal (^) ectopic eyes. It is unknown whether these (^) ectopic eyes are (^) functional, and wheth- er the axons of the (^) photoreceptors innervate

the correct domains of the brain, that is, the

lamina and the dorsal deuterocerebrum, re-

spectively (19).^ Initial evidence^ suggests^ that the (^) photoreceptors in the (^) ectopic eyes are electrically active^ upon illumination^ (20). Role of (^) eyeless in (^) eye morphogenesis. The (^) reported findings indicate that (^) ey is the master control (^) gene for (^) eye morphogenesis, because it can induce (^) ectopic eye structures in at least the (^) imaginal discs of the head and thoracic (^) segments. The (^) expression of (^) ey

switches on the (^) eye developmental pathway that involves several thousand (^) genes. The number of (^) genes required for (^) eye morpho- genesis can^ roughly be estimated^ on the basis of the (^) frequency of enhancer detection lines that show (^) reporter gene expression in the eye imaginal discs^ posterior to^ the^ morpho- genetic furrow^ during eye differentiation.^ Be- cause (^) approximately 15 percent of a^ large sample of enhancer detector^ lines fall into this (^) category (21), and (^) assuming that the Drosophila genome contains^ at least^ 17, genes (22),^ we^ estimate^ that more than 2500 genes are involved in^ eye morphogenesis. Our results (^) suggest that all of these (^) genes are under the direct or indirect control^ of^ ey, which is at the (^) top of the (^) regulatory cascade or (^) hierarchy. The (^) ey gene is (^) expressed first and controls a set of subordinate (^) regulatory genes, including sine^ oculis,^ another ho- meobox-containing gene (23).^ Subsequent- ly, genes that^ influence cell-cell^ interactions and (^) signal transduction must be (^) regulated and, finally, the structural^ genes like^ rhodop- sin, (^) crystallin, and transducin^ must be^ ex- pressed. The lower^ part of this^ cascade,^ in- cluding signal transduction^ pathways, has been elucidated to a (^) large extent (^) (24), but

the upper part, and which of these interac-

Fig 5.^ The^ ectopic expression^ of^ mouse Pax-

cDNA under the control of GAL4 induces the for-

mation of ectopic eyes (26). The scanning electron
micrograph shows^ a^ close-up^ of induced^ eye^ fac-
ets on a leg. Ommatidial arrays and interomma-
tidial bristles very similar to the ectopic eye struc-
tures induced by the Drosophila gene (Fig. 3) were
formed (30). In^ both cases^ the same GAL4^ line

E132 was used.

tions are^ direct, remain^ to be determined.

However, (^) ey may not^ only control^ the^ initial steps of^ eye morphogenesis, but^ also,^ as^ sug- gested from^ the^ expression pattern, it^ may control later (^) steps. Thus, the^ same^ transcrip- tional (^) regulator may be used at consecutive steps of^ morphogenesis. This could^ be the consequence of the conservative^ mode^ of evolution (^) whereby the same master control gene is used^ repeatedly to^ integrate^ new tar- get genes into the^ eye developmental path- way. In addition^ to^ eye morphogenesis, ey controls other functions in the^ developing nervous (^) system, because null mutations are lethal, and the loss of^ eye structures^ alone^ is not the cause of (^) lethality. The induction of (^) ectopic eyes in (^) Drosoph- ila is reminiscent of the classical (^) experiments of (^) Spemann (25) in which he induced ec- topic eyes by transplanting the^ primordia of the (^) optic cup to (^) ectopic sites in (^) amphibian embryos. Our^ experiments extend these^ ob- servations and (^) identify the (^) gene that is nec- essary and sufficient to^ induce^ ectopic eyes at least in (^) imaginal discs. In the (^) mouse, (^) Sey is expressed at each^ step of the induction^ pro- cess; first in the^ optic cup, then^ in^ the^ lens, and (^) finally in the (^) cornea, which (^) implies that Sey may be the master^ control^ gene in the mouse (^) eye induction (^) process (4). The transformation of (^) antennal, leg, and wing tissue into^ eye structures^ by ey induc- tion indicates (^) that (^) ey is a (^) homeotic (^) gene. In contrast to the classic homeotic (^) genes of the Antennapedia and Bithorax^ Complexes, hy- pomorphic loss-of-function^ mutations^ in^ ey

do not lead to homeotic transformation, but

rather, (^) they result^ in^ the^ loss of^ eye struc- tures. (^) However, (^) targeted ectopic ey expres- sion induces homeotic transformations sim-

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ilar to those (^) observed in (^) gain-of-function mutations of classic homeotic (^) genes, like

Antp. Therefore,^ ey represents a class of^ ho- meotic master control (^) genes different from Antp. Gain-of-function mutants with^ pheno- types corresponding to those^ obtained^ in^ our targeted gene expression experiments have not been discovered (^) previously. The (^) high degree of (^) sequence conserva-

tion between the human, the mouse, and the

Drosophila genes, the^ similarity of the^ phe- notypes of^ Aniridia, Sey, and^ ey, and the similarity of the^ expression patterns suggest- ed to (^) us that (^) ey might be a master control gene for^ eye morphogenesis that is shared^ by

vertebrates and invertebrates (3). Because

we also found (^) homologous genes in ascid- ians, (^) cephalopods, and nemerteans we^ pro- pose that^ ey function is universal^ among metazoa. In order to test whether the mouse gene can substitute for the^ Drosophila gene, we also used the (^) mouse (^) Sey gene for (^) targeted expression in^ Drosophila. Similar to^ the re- sults obtained for the (^) Drosophila ey gene, the mouse (^) gene Sey can also induce the forma- tion of (^) ectopic eye structures (^) (Fig. 5) (26). As (^) expected, the (^) ectopic eye structures formed contain (^) Drosophila-type ommatidia and not mouse (^) eye structures. Previously, the function of other mouse homeobox (^) genes has been demonstrated in Drosophila with the use of heat inducible vectors (^) (27). In the case of (^) HoxB6, Dro- sophila (^) legs were induced^ in^ place of the antennae (^) (27). (^) Obviously, the (^) responses, but not the (^) transcriptional regulator, are species-specific. The observation that mammals and in- sects, which have evolved^ separately for more than 500 million (^) years, share the same master control (^) gene for (^) eye morphogenesis indicates that the (^) genetic control mecha- nisms of (^) development are much more uni- versal than (^) anticipated. It will be informa- tive to (^) compare the (^) regulatory cascade re- quired to form a^ Drosophila compound eye with that of a mouse (^) eye, to find out what the differences (^) are, and to determine how many new^ genes have been recruited into these (^) developmental pathways in the course of evolution.

REFERENCES AND NOTES

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  6. The (^) expression pattern of (^) ey was (^) analyzed by stain- ing of^ the different mutant^ eye-antennal discs^ with^ a polyclonal rat^ ey antibody and a fluorescein-labeled secondary antibody. The mutants have been de-

scribed in D. L. (^) Lindsley and G. G. Zimm, The Ge- nome of (^) Drosophila melanogaster (Academic Press, New (^) York, 1992), p. 1133.

  1. W. (^) McGinnis and R. Krumlauf, Cell 68, (^283) (1992); J. Botas, Cur.^ Biol.^ 5, 1015 (1993); W.^ J.^ Gehring, M. Affolter, T.^ B0rglin, Annu. Rev. Biochem.^ 63, 487 (1994).
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  7. C. J. O'Kane and W. J. (^) Gehring, Proc. Natl. Acad. Sci. U.S.A.^ 84, 9123 (1987); H.^ Bellen^ et^ al., Genes Dev. (^) 3, 1288 (1989); C. Wilson et (^) al., ibid. (^) 3, 1301 (1989); E. Bier et^ al., ibid., p. 1273.
  8. GAL4-crosses were established at 25°C on standard medium. The GAL4 lines were crossed to a number of UAS-ey lines and most of these crosses^ led to^ embry- onic or (^) early larval (^) lethality. This outcome is (^) probably the result of the (^) ectopic expression of (^) ey in various tissues (^) during embryogenesis because the GAL lines start to (^) express GAL4 (^) during embryonic stages. In crosses with the (^) MS941, (^) p339, and E132 (^) lines, transheterozygote adults^ were^ recovered.^ For MS941, almost no (^) lethality was observed, whereas for (^) p339 and E132, a substantial number of dead embryos or^ larvae^ were noted. For line^ E132,^ virtually only females were^ obtained^ because most males died during the^ early phases of^ development. This result may be^ explained by the^ dependence of the^ lethality on the level of transactivation of (^) ey by GAL4. In the El (^32) line, the enhancer detector construct is inserted into the X chromosome and (^) therefore, is (^) dosage- compensated in^ males. As a^ consequence, the^ trans- heterozygous males^ produce twice as much GAL activity as the^ females, and die^ during larval^ stages, whereas the females survive. (^) Thus, all cuticles shown are derived from females.
  9. The (^) full-length embryonic cDNA was reconstructed in a (^) Bluescript KS+ backbone from three Eco (^) RI (^) frag- ments. The (^) full-length embryonic cDNA (^) begins with a Hind (^) III site (^) [at position 45 in the (^) published sequence (3)] and ends^ with^ an Xba site^ (constructed by insert- ing Xba linkers^ in^ the Msl site at^ position 2741). The cDNA was inserted as an Xho (^) I-Xba fragment into the GAL UAS vector (^) [pUAST (11)]. This construction results in an oriented insertion in which the cDNA is preceeded at the 5' end^ by five^ optimized GAL4 bind- ing sites, an^ hsp70TATA^ box, the^ transcriptional start, and the cDNA is followed at the 3' end (^) by the (^) SV intron and (^) polyadenylation site. A (^) y ac w stock was transformed as described (^) [G. M. Rubin and A. C. Spradling, Science^ 218,^348 (1982)]. A^ total^ of^13 independent pUAST-ey strains were^ analyzed. The heat-inducible construct was made (^) by inserting the embryonic cDNA into the heat-shock^ Casper vector [V. Pirrotta,^ in A^ Survey of^ Molecular^ Cloning Vectors and Their (^) Uses, R. (^) L. (^) Rodriguez and D. T. Denhardt (Buttesworth, Boston and^ London, 1988), p. 437].
  10. G. M. (^) Edelmann, Annu. Rev. Cell Biol. (^) 2, (^81) (1986).
  11. (^) Wild-type and (^) ectopic eyes were dissected and fixed for 30 minutes on ice in a (^) cacodylate-buffered glutaraldehyde-oxmiumtetroxide fixation mixture. After (^) postfixation in (^) cacodylate-buffered osmi- umtetroxide, the tissue was^ dehydrated through an ethanol series and embedded in (^) Spurr medium. One-micrometer sections were cut and stained with (^) staining solution (^) (equal volumes of (^2) percent Azur II^ and (^2) percent disodiumtetraborate to (^2) per- cent (^) methylene blue). After (^) drying they were

mounted with (^) Depex.

  1. (^) Staining of (^) imaginal discs with ELAV antibodies was performed according to^ S.^ Robinow^ and^ K. White^ [J. Neurobiol. 22,443 (^) (1991)]. Afluorescein-conjugated secondary rat^ antibody (Cappel) was^ used.^ Analysis was done on a Zeiss (^) Axiophot microscope equipped for (^) epifluorescence.
  2. I. A. (^) Meinertzhagen and T. E. (^) Hanson, in The Devel- opment of^ Drosophila melangoaster, M.^ Bate and^ A. Martinez-Arias, Eds.^ (Cold Spring Harbor (^) Laboratory Press, Cold (^) Spring Harbor, NY, (^) 1993), chap. 24, (^) p.
  3. P. (^) Callaerts, G. (^) Halder, W. (^) Gehring, in (^) preparation.
  4. G. M. (^) Rubin, personal communication.
  5. L. M. (^) Hall, P. J. (^) Mason, P. (^) Spierer, J. Mol. Biol. (^) 169, (^83) (1983); B. (^) Bossy, L. M. C. (^) Hall, P. (^) Spierer, EMBO 3, (^2537) (1984); J. Gausz, L. M. C. Hall, A. (^) Spierer, P. Spierer, Genetics^ 112,^65 (1986); J.^ Mirkovitch,^ P. Spierer, U. K.^ Laemmli,^ J. Mol. Biol.^ 190,255^ (1986). The (^) haploid genome size of (^) Drosophila is estimated to be 165,000 kb. The (^) gene density can be estimat- ed from the number of (^) transcription units detected on a chromosome walk of 315 kb and amounts to approximately one^ gene per 7 kb.^ After^ subtraction of (^22) percent of satellite and other (^) repetitive DNA, we obtain a value of (^) approximately 17,000 genes per haploid genome. This is^ certainly an^ underestimate because not all (^) transcripts from this (^) region have been (^) detected, and on the basis of (^) experience with genomic (^) sequencing of^ yeast chromosomes, a con- siderably (^) larger value is to be^ expected.
  6. B. N. R. (^) Cheyetteetal., Neuron (^) 12,977 (^) (1994); M. A. Serikaku and J. E. (^) O'Tousa, Genetics 138, 1137 (1994).
  7. (^) B. Dickson and E. Hafen, in The (^) Development of Drosophila melangoaster, M.^ Bate and^ A.^ Martinez- Arias, Eds. (^) (Cold Spring Harbor (^) Laboratory Press, Cold (^) Spring Harbor, NY, 1993), chap. 23, p. 1327; S. L. (^) Zipursky and G. M. (^) Rubin, Annu. Rev. Neurosci. 17, 373(1994).
  8. H. (^) Spemann, Embryonic (^) Development and Induc- tion (^) (Yale (^) University Press, New (^) Haven, CT, (^) 1938).
  9. The (^) full-length mouse Pax-6 cDNA (^) (a gift of C. Walther and P. (^) Gruss) was cloned as a Not (^) I-Xho fragment in the GAL-UAS vector (^) [pUAST (11)]. Flies were trans- formed as described in (^) (15). To (^) ectopically induce the mouse Pax-6 (^) gene, the UAS-Pax-6 transformant lines were crossed to the E1 32 GAL4 (^) expressing line as for the (^) Drosophila gene (14). The (^) figure shows an ectopic eye on a^ second^ leg of a male.
  10. J. Malicki (^) etal., Cell (^) 63, 961 (1990); J. J. (^) Zhao, R. A. Lazzarini, L.^ Pick, Genes Dev.^ 7, 343 (1993); D. Bachiller et (^) al., EMBO J. (^) 13, 1930 (1994).
  11. (^) p-Galactosidase (^) staining was (^) performed as de- scribed in M. (^) Ashburner, (^) Drosophila, A (^) Laboratory Manual (^) (Cold (^) Spring Harbor (^) Laboratory Press, Cold Spring Harbor, NY,^ 1989), protocol 77.
  12. For cuticle (^) preparations, adults were dissected in phosphate-buffered saline, mounted in^ Hoyer's or Faure's (^) mounting medium, and (^) immediately photo- graphed to avoid diffusion and^ bleaching of^ eye (^) pig- ments.
  13. For (^) scanning electron (^) microscopy, freshly hatched flies were narcotized and immersed in (^70) percent acetone. After critical (^) point (^) drying, they were mount- ed and coated with (^) gold. The (^) specimens were ob- served with a Hitachi S-800 field emission electron microscope at 6^ kV.
  14. We thank the (^) following colleagues for (^) fly stocks, anti- bodies, and^ plasmid vectors: S.^ Benzer, A.^ Brand, C. Goodman, P.^ Gruss, E. Hafen, F.^ Jimenez, W.^ E. Kalisch, C.^ O'Kane, V.^ Pirrotta, C.^ Walther, and L. Zipursky; C. O'Kane and his^ group for^ the^ support of one of us (^) during his (^) stay in the O'Kane (^) laboratory (G.H.); E.^ Hafen and collaborators for advice on^ histo- logical and^ antibody staining techniques (to P.C.); and the (^) Interdepartmental Electron (^) Microscopy Service for (^) assistance, in (^) particular A. Hefti and U. (^) Sauder; E. Marquardt-Wenger for^ processing the^ manuscript; and M. (^) Affolter, W. (^) Keegan, and F. Loosli for critical discussions. (^) Supported by the (^) Collen Foundation of Leuven, Belgium (P.C.), the Kantons of^ Basel, and the Swiss National Science Foundation. (^1) February 1995; (^) accepted 24 February 1995

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