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RESEARCH ARTICLE
659
Development 134, 659-667 (2007) doi:10.1242/dev.02786
Dorsal-ventral midline signaling in the developing
Drosophila eye
Atsushi Sato and Andrew Tomlinson*
Boundaries between different cell types play key roles in many developmental patterning processes. They can be established by
various mechanisms, and signaling between the different cell types can occur in a number of ways. One mechanism of crossboundary signaling is controlled by the Notch (N)-modifying protein Fringe (Fng). At the Drosophila wing dorsal-ventral (D-V)
border, the mechanism by which an Fng+-Fng– interface controls local N activation has been well characterized. A similar Nactivating Fng+-Fng– interface has also been described at the D-V border of the fly eye, but the mechanisms that establish and
regulate it are different from those in the wing. Here we describe the ventral role of the Sloppy-paired (Slp) transcription factor,
and its interactions with dorsally expressed Iroquois (Iro) transcription factors in the regulation of signaling about the Fng+-Fng–
interface in the developing eye. The two transcription factors are mutually repressive and initially abut at the D-V midline. However,
N signaling at the interface downregulates Slp expression, and a gap opens between the two expression domains in which Serrate
(Ser, an N ligand) is upregulated.
INTRODUCTION
Development is a stepwise process in which new information is
generated through the interactions of previously specified cell types.
The place where different cell types abut often defines the position
from which secreted molecules are released, and these signals then
direct further elaboration of patterning (Lawrence and Struhl, 1996).
The cell types at the interface not only define the position from
which the signal will be released, but they also carry the molecular
information that directs which type of signal will be expressed, and
the modes of response of the cells. Interfaces between different cell
types can be established by a number of mechanisms. In the fly
wing, the dorsal-ventral (D-V) boundary is established by a
compartment mechanism, whereby cells on either side of the border
are clonally isolated from one another: dorsal cells express the
Apterous (Ap) transcription factor and share a lineage ancestry
isolated from the ventral cells that do not express Ap (Blair et al.,
1994; Diaz-Benjumea and Cohen, 1993). Differential adhesion
between the two cell types leads to the formation of a long interface
(the D-V border). Borders between distinct cell types can be
generated by non-compartment mechanisms. For example, in the
vertebrate neural tube, Sonic Hedgehog released from the ventral
floorplate diffuses dorsally to elicit the expression of an array of
different transcription factors, each expression domain depending
on its distance from the source (Jessell, 2000). This first step
establishes a relatively crude array of expression domains with many
cells expressing more than one transcription factor. The transcription
factors, however, are autoregulatory and mutually repressive, and if
a cell initially expresses two, the action of one will force the
extinction of the other. As with the compartment mechanism, the
transcription factors also provide a miscibility code, such that likecells mix and unlike-cells repel, and sharply defined interfaces are
formed.
Department of Genetics and Development, College of Physicians and Surgeons,
Columbia University, 701 West 168th St, HHSC 1120, New York, NY 10032, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 14 December 2006
There is great variability in the types of signaling that can occur
at interfaces. A particularly important form is that regulated by
Fringe (Fng), a glycocyltransferase that modifies the receptor
Notch (N) and thereby modulates its ligand sensitivities (Bruckner
et al., 2000; Moloney et al., 2000; Munro and Freeman, 2000).
The N/Fng-mediated signaling was first described at the D-V
border of the fly wing (Klein and Arias, 1998; Panin et al., 1997)
and has since been described elsewhere in fly and vertebrate
patterning (Haines and Irvine, 2003; Irvine, 1999). Once a Fng+Fng– border is established, a dynamic interaction between N and
its ligands leads to high-level ligand expression and consequential
upregulation of N activity.
A Fng+-Fng– boundary also forms at the D-V midline of the fly
eye and the resulting N signaling activates a number of genes needed
for growth and patterning of the eye (Cho and Choi, 1998;
Dominguez and de Celis, 1998; Papayannopoulos et al., 1998). The
D-V midlines of the eye and wing display a number of major
differences. First, the D-V domains of the eye do not appear to be
generated by a compartment mechanism as clones can violate the DV midline (Becker, 1957). Second, in the wing, Fng is expressed
dorsally (under Ap transcriptional control), but in the eye it is
expressed ventrally [repressed dorsally by Iroquois (Iro)
transcription factors] (Cho and Choi, 1998; Dominguez and de
Celis, 1998; Irvine and Wieschaus, 1994; Papayannopoulos et al.,
1998). Thus, the eye and wing both use the Fng/N signaling
mechanism at the D-V midlines, but the mechanisms by which the
interfaces are established are different.
Sloppy-paired 1 and 2 (Slp1 and Slp2) are homologous Forkhead
transcription factors that have extensive and frequently redundant
roles in embryonic patterning (Grossniklaus et al., 1992). We
describe here the ventral-specific expression of Slp proteins in the
developing eye. We show that Slp and Iro proteins are mutually
repressive transcription factors expressed in the ventral and dorsal
domains of the developing eye, respectively. Initially, the two
domains directly abut at the center of the eye disc, but as
development proceeds a gap opens up between them. This gap is
caused by the N-induced downregulation of slp, which facilitates the
upregulation of Serrate, one of the N ligands.
DEVELOPMENT
KEY WORDS: Drosophila, Eye, Sloppy-paired, Dorsoventral signaling
RESEARCH ARTICLE
Development 134 (4)
MATERIALS AND METHODS
Histology
Fly stocks
Antibodies used were rat anti-Mirr (Yang et al., 1999) (1:2000), rat anti-Ser
(Papayannopoulos et al., 1998) (1:1000), mouse anti-Dl (DSHB, 1:600),
rabbit anti-LacZ (Cappel, 1:2000), mouse anti-␤-galactosidase (Promega,
1:500), anti-rabbit IgG Alexa Fluor 488-conjugated (Molecular Probes,
1:1000), anti-mouse IgG Alexa Fluor 488-conjugated (Molecular Probes,
1:1000), anti-rabbit IgG Cy3-conjugated (Jackson, 1:1000), anti-mouse IgG
Cy3-conjugated (Jackson, 1:1000), anti-rat IgG Cy3-conjugated (Jackson,
1:1000), anti-mouse IgG Cy5-conjugated (Jackson, 1:500) and antidigoxigenin alkaline phosphatase-conjugated (Roche, 1:2000). Standard
methods were used for antibody staining. slp1, slp2 (Grossniklaus et al.,
1992), mirr (McNeill et al., 1997) and fng (Irvine and Wieschaus, 1994)
cDNAs were used for in situ hybridization following Curtiss and Mlodzik
(Curtiss and Mlodzik, 2000) and/or Sato et al. (Sato et al., 1999).
Fly strains used are: yw, slp105965 (slp1-lacZ; Bloomington Stock Center),
mirrF7 (mirr-lacZ) (McNeill et al., 1997), eyg-Gal4 (Wehrli and Tomlinson,
1998), Tub>Gal80, y+>Gal4 (gift from Gary Struhl), UAS-slp1 (gift from
Gary Struhl), UAS-slp2 (Riechmann et al., 1997), UAS-mirr (Cavodeassi
et al., 2000), UAS-Nintra (Struhl and Adachi, 1998), UAS-GFP
(Bloomington Stock Center), iroDFM3 (Gomez-Skarmeta et al., 1996), UASP35 (Hay et al., 1994), Nts (Shellenbarger and Mohler, 1978) and hs-N-GV
(Chung and Struhl, 2001). Clones were made using slpS37A FRT40A, ubiGFP, FRT40A (Bloomington Stock Center), hs-GFP M(2) w+30C FRT40A
(gift from Gary Struhl), iroDFM3 FRT2A (Tomlinson, 2003) and hs-GFP
M(3)i55 FRT2A chromosomes (gift from Gary Struhl). We also made
slpS37A mutant clones with UAS-P35 using MARCM methods (Lee and
Luo, 1999). Clones were induced at 48 or 72 hours before dissection. Nts
flies were grown at 18°C and shifted to 31°C and left for 24 hours before
dissection.
slpAVm is an insertion line of a UAS construct at the slp2 locus. The
insertion site was determined by inverse PCR and sequencing. slpS37A was
made by imprecise P-element excision from slpAVm, and screened by
complementation with slp105965. Break points of the deletion were
determined by PCR and sequencing. Part of the P-element was not deleted.
This sequence of this lesion is available at GenBank (Accession No.
EF141189).
RESULTS
An insertion (slpAvm) of a construct carrying a mini-w+ selectable
marker (transformed for other purposes) showed a ventral-anterior
pigmentation pattern in adult eyes (Fig. 1A). This expression pattern
suggested that the ‘trapped’ gene was expressed exclusively in the
ventral region of the developing eye. Inverse PCR identified the slp
locus as the site of insertion (Fig. 1B). There are two transcription
units in the slp locus (slp1 and slp2) and in situ hybridization to eye
Fig. 1. Expression patterns, mutant phenotypes and genomic organization of slp. (A) Adult eye of the slp2 insertion line (slp2AVm) shows
pigmentation in the anterior/ventral domain of the eye. (B) Genomic map of the slp locus showing the site of insertion of slp2AVm and the deletion
induced (slpS37A, dotted lines). (C) slp-lacZ expression in the second-instar eye disc. (D) mirr-lacZ expression in the second instar. (E) Double staining
of slp-lacZ (red) and Mirr (green) of second-instar eye disc. The two domains abut in the center of the disc. (F) Double staining of slp-lacZ (red) and
Mirr (green) of third-instar head disc. The white arrowhead indicates the gap between the slp and Mirr expression domains. Arrows indicate slp
expression in the dorsal regions of the extreme anterior of the eye disc, and in the posterior dorsal (relative to the eye) regions of the antennal disc.
(G-I) Survival of slpS37A mutant clones (black) in third-instar eye discs, induced at different times of development. Yellow arrowhead indicates
morphogenetic furrow (MF). (G) When clones are induced 0-24 hours AEL, twin spots (arrow) are readily evident in the ventral regions, but mutant
clones are rarely observed. (H) Clones induced 24-48 hours AEL do not survive ventrally ahead of the MF (arrow points to a twin spot), but can be
observed behind (white arrowheads), but are rounded with smooth borders. (I) Clones induced 48-72 hours AEL. slpS37A mutant clones (white
arrowheads) survive ahead of the MF but show smooth borders. In all images, dorsal is up and posterior is left.
DEVELOPMENT
660
discs showed that the two were co-expressed (not shown) in the
same pattern as the slp1-lacZ line (slp105965). slp-lacZ thus appears
to be a faithful reporter of transcription of the slp locus in the
developing eye. slp-lacZ expression was observed in the ventral
domain of the second-instar eye disc, extending into the posterior
portion of the antennal disc (Fig. 1E), and in the anterior/ventral
regions of late-third-instar discs (Fig. 1F), and also in dorsal regions
of the extreme anterior of the eye disc and in the posterior dorsal
(relative to the eye disc) regions of the antennal disc (arrows in Fig.
1F).
Eye discs were double-stained for transcriptional activity of the
iro complex (dorsally expressed) and the slp complex (ventrally
expressed) to determine the spatial relationship of the two expression
domains. In second-instar eye discs, mirror (mirr, one of the three
iro genes) transcription was exclusively dorsal (Fig. 1D,E) whereas
slp was exclusively ventral (Fig. 1C,E), and the two expression
domains directly abutted (with no overlap) in the medial region of
the eye disc (Fig. 1E). In the third larval instar the morphogenetic
furrow (MF) begins to progress across the presumptive retina, and
slp and mirr transcription was extinguished in post-furrow regions
as evidenced by in situ hybridization (not shown). However, lacZ
reporters from both genes showed persistent expression posterior to
the MF, which is likely to result from perdurance of the long-lived
lacZ gene product (Fig. 1F). Ahead of the furrow mirr was still
exclusively dorsal, and slp ventral, but now the two expression
domains did not abut; instead, a gap opened that appeared to be
generated by loss of slp expression in the region close to the D-V
midline (white arrowhead in Fig. 1F).
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661
Generation of a slp1, slp2 mutant
slp1 and slp2 are functionally redundantly elsewhere in Drosophila
development (Grossniklaus et al., 1992), and as transcripts from both
genes were expressed identically in the developing eye, both needed
to be removed for an effective loss-of-function analysis to be
performed. The only existing lesion that removes both transcripts
(without affecting other loci) is on a CyO balancer chromosome
(Grossniklaus et al., 1992; Grumbling and Strelets, 2006) that was
not useful to us. We therefore generated a new deficiency mutation
(slpS37A) by mobilization of the slpAvm insertion. Molecular
characterization of slpS37A showed a deletion that removes the slp1
transcription unit and all intervening sequence to 16 bp downstream
of the slp2 transcriptional start site (Fig. 1B). Thus, although the slp2
coding sequence is intact, the promoter sequence is removed. To test
whether the lesion is effectively null for both transcripts it was
crossed to a slp⌬34B deficiency and a ‘null’ embryonic phenotype of
fused denticle belts was observed (not shown) (Grossniklaus et al.,
1992).
slp phenotypes in the ventral eye
slpS37A mutants are embryonic lethal, and to assess Slp function in the
developing eye, clones were induced at various developmental stages
and examined in late-third-instar discs. Regardless of the time of
induction, ventral clones showed significant differences in appearance
and frequency to their dorsal counterparts (Fig. 1G-I). Clones induced
at 0-24 hours after egg lying (AEL), although frequently present
dorsally, were largely absent from the ventral domain (Fig. 1G).
Clones induced at 24-48 hours AEL occurred in the ventral region of
Fig. 2. The reciprocal effects of ectopic expression or loss of Slp and Iro. (A-A⬙) mirr-lacZ (red) is repressed by ectopic slp (green, arrow). Inset
in A⬙ is higher magnification showing the autonomous repression of mirr-lacZ (arrows mark diminishing staining inside dorsal slp). (B-B⬙) slp-lacZ
(red) is ectopically expressed in the inner regions of iro mutant clone (loss of green). Note that the ectopic slp-lacZ evident dorsal to the arrowed
clone is an outgrowth of eye tissue into the head capsule. Why slp is non-autonomously induced in such outgrowths is unclear. (C-C⬙) slp-lacZ (red)
is repressed by ectopic mirr (green, arrow). Inset in C⬙ shows the non-autonomous effects on slp-lacZ. Immediately surrounding the clone staining is
absent, and surrounding this is a ring of cells showing decreased expression. (A⵮, B⵮, C⵮) Schematic summaries of the effects observed.
DEVELOPMENT
Dorsoventral signaling in the fly eye
RESEARCH ARTICLE
the eye discs but differed from their dorsal counterparts; they were
significantly less frequent, and were found predominantly behind the
MF (Fig. 1H). Cells in the posterior regions of the eye are taken out of
the cell cycle and ‘frozen’ into the ommatidial lattice earlier than their
anterior counterparts. Thus, the absence of anterior/ventral clones
suggests that perduring slp gene product allows mutant cells to survive
for a time (the posterior clones; white arrowheads in Fig. 1H), but that
more extensive proliferation leads to further dilution of the gene
product, followed by loss of the cells. Surviving ventral clones were
also rounded compared with their dorsal counterparts (Fig. 1H),
suggesting an adhesive difference between wild-type and slp ventral
cells. Further support for the perdurance phenomenon came from
clones induced at 48-72 hours AEL; now they occurred on both sides
of the MF, but still showed smoother borders than their dorsal
counterparts (white arrowheads in Fig. 1I). These data suggest that Slp
function is required for the growth/survival/persistence of cells in the
ventral region of the disc. In an attempt to generate large slp patches,
we generated slp clones in a Minute background, but these clones were
not appreciably different from those generated in a wild-type
background (not shown). To determine whether the slp cells were
removed by apoptosis, slpS37A clones were induced in the presence of
the baculovirus apoptotic caspase inhibitor P35 (Hay et al., 1994).
Again, these clones were similar to those induced in wild-type tissues,
and thus the under-representation of ventral slpS37A cells does not
appear to result from programmed cell death.
Development 134 (4)
Slp and Iro are mutually repressive transcription
factors
Since Slp and Iro are transcription factors expressed in abutting
domains of the second-instar disc (Fig. 1E), we examined the ability
of one to regulate the expression of the other. When slp1 was
ectopically expressed in dorsal clonal patches (marked by the coexpression of GFP), a correlating cell-autonomous suppression of
mirr transcription was observed (Fig. 2A). Some of the Slpexpressing cells showed residual mirr-lacZ expression (inset in Fig.
2A⬙), but this represented significantly reduced staining, suggesting
that these cells are in the process of extinguishing mirr expression.
Furthermore, the lacZ gene product has long perdurance. Thus, Slp
appears to be an effective suppressor of iro gene expression. When
mutant clones for all three genes of the iro gene complex (iroDFM3)
were induced in dorsal regions, slp transcription was coincidentally
upregulated (Fig. 2B). However, slp was typically expressed in the
center of such clones and not at their peripheries (Fig. 2B). This
observation suggests: (1) that Iro expression normally functions to
repress dorsal expression of slp; and (2) that the creation of an Iro+Iro– interface generates an influence that locally suppresses slp
expression (the absence of slp expression at the clone periphery).
When mirr was ectopically expressed in ventral clones, a
correlating suppression of endogenous slp transcription occurred
(Fig. 2C). Again, a non-autonomous effect occurred and slp
expression in the cells surrounding the clone was also
Fig. 3. (A,B) Double staining of fng in
situ hybridization (red) and mirr-lacZ
expression (green) in second (A) and
mid-third (B) instars. Arrowhead in B
indicates the interface between fng
and mirr expression. (B⬘) Arrows
show the potential Fng+-Fng–
interface in the ventral anterior
regions of the eye disc as evidenced
by fng expression. (C-E⬙) Regulation
of fng expression by Slp and Iro as
evidenced by fng in situ hybridization.
(C) Clones ectopically expressing slp
(green, arrows) show upregulation of
fng transcription (red). (D) Clones
ectopically expressing mirr (green,
arrow) downregulate fng
transcription (red). (E) When slp and
mirr are co-expressed ventrally, fng
transcription (red) is repressed in
ventral (arrowhead), but not
upregulated in dorsal (arrow) regions.
DEVELOPMENT
662
downregulated. Immediately surrounding the clone was a region
devoid of slp-lacZ expression, and surrounding that was a band of
cells with reduced expression (inset in Fig. 2C⬙). Thus, Iro is an
effective repressor of slp expression. Furthermore, this experiment
provides another example of an Iro+-Iro– interface generating an
influence that locally suppresses slp expression.
The sub-viability of ventral slp clones prevented an effective
assessment of any role that Slp might play in suppression of ventral
iro expression.
These above experiments indicate: (1) that Slp and Iro are
mutually repressive transcription factors; (2) that Iro actively
suppresses slp expression in the dorsal domain; and (3) that an Iro+Iro– interface generates an slp-repressing influence, which we show
below to be N activity.
The roles of Iro and Slp in regulating fng
expression
Fng is expressed exclusively in ventral eye disc cells (red in Fig. 3AB), and at the D-V midline a Fng+-Fng– interface is formed
(arrowhead in Fig. 3B). Hitherto, Iro has been shown to repress fng
transcription dorsally (Cho and Choi, 1998; Dominguez and de
Celis, 1998; Papayannopoulos et al., 1998). However, when slp is
ectopically expressed in dorsal cells, an upregulation of fng
transcription occurs (arrows in Fig. 3C). Correspondingly, when iro
is clonally expressed ectopically in the ventral territory, a
downregulation of fng transcription occurs (arrow in Fig. 3D). This
raised the question of whether Iro prevented dorsal fng expression
indirectly, by repressing slp expression. When slp and mirr were coexpressed in clonal patches (both dorsally and ventrally), they
behaved as mirr alone; there was no dorsal fng upregulation (arrow
in Fig. 3E) and there was repression of ventral fng expression
(arrowhead in Fig. 3E). Furthermore, we note that surviving ventral
slp mutant clones do not show phenotypes typical of Fng+-Fng–
interfaces (not shown), which would have arisen if Slp activated
ventral fng expression. Thus, it appears that Slp does not promote
the ventral expression of fng.
Notch activity suppresses slp expression
In second-instar eye discs, the Slp and Iro domains directly abut at
the midline of the disc (Fig. 1E), and yet by the late third instar a gap
opens between them (Fig. 1F). Furthermore, in the clonal
experiments described above, whenever an Iro+-Iro– interface was
generated, a local downregulation of slp transcription was observed
RESEARCH ARTICLE
663
(Fig. 2B-C). Thus, wherever we observed an Iro+-Iro– interface,
whether at the normal midline or in clonal experiments, there was a
local downregulation of slp expression. Since Iro+-Iro– interfaces
establish Fng+-Fng– interfaces (which lead to local N activation), we
wondered whether N was the slp-repressing signal.
When a constitutively activated form of the N protein (Nintra)
(Struhl et al., 1993) was expressed in the ventral regions of the eye
discs, a corresponding downregulation of slp expression occurred
(Fig. 4A). Thus N activation appears sufficient to repress slp
expression, suggesting that the gap that opens up between the Iro and
Slp domains in the third-instar disc is caused by the local N
activation at the midline interface. If this were true, then removal of
N function should prevent the formation of the gap.
To test whether endogenous N gene function was required for the
downregulation of slp, Nts mutant (Shellenbarger and Mohler, 1978)
larvae were grown at the permissive temperature until early third
instar and then shifted to 31°C for 24 hours before dissection and
staining. Such discs showed a complete absence of slp repression,
and the Slp and Iro domains abutted along the length of the midline
interface (Fig. 4C). The disc shown in Fig. 4C (Nts/Y) was treated
identically to the control shown in Fig. 4B (Nts/+), but has a more
immature appearance because N also functions in MF progression
(Baonza and Freeman, 2001).
These experiments show that ectopic N activity suppresses slp,
and that loss of endogenous N activity prevents the normal
downregulation of slp expression. Thus, we infer that N activity is
the influence present at Iro+-Iro– interfaces that downregulates slp
expression.
The regulation of Dl and Ser expression
In the eye, Iro+-Iro– interfaces generate corresponding Fng+-Fng–
interfaces, which regulate the activity of the two N ligands Delta (Dl)
and Serrate (Ser), leading to a local upregulation of N activity. One
of the outputs of the N signaling is a local increase in the expression
of its ligands, which thereby further strengthens the N activity (de
Celis and Bray, 1997).
In the second-instar eye disc, Dl was ubiquitously expressed (red
in Fig. 5A), whereas Ser was expressed in the region of the D-V
border (arrow in Fig. 5A) (Cho and Choi, 1998; Dominguez and de
Celis, 1998; Papayannopoulos et al., 1998). In the third larval instar,
N ligand upregulation occurred in two positions – one associated
with the Iro+-Iro– interface, and the other not. These will be
described below.
Fig. 4. N activation downregulates
slp expression. (A-A⬙) Clones of
Nintra (green, arrows) show
downregulation of slp-lacZ expression
(red). (B,C) The effects of a 24-hour
temperature shift on Nts/+ control
discs (B) and Nts/Y experimental discs
(C) stained for slp-lacZ (green) and
Mirr (red). In the control, the gap
(arrowhead) forms normally, but in
the Nts/Y, slp transcription is
unaffected and the gap fails to form.
DEVELOPMENT
Dorsoventral signaling in the fly eye
664
RESEARCH ARTICLE
Development 134 (4)
Fig. 5. N activity and
expression of its ligands in
second and third-instar eye
discs. (A-A⬙) In late-secondinstar eye discs, Ser (arrow,
green) is expressed in a broad
swath about the D-V midline,
whereas Dl (red) expression is
more general. (B) Mid-thirdinstar N activation evidenced by
N-GV/UAS-lacZ staining. Ahead
of the wave front, strong N
activity is in a line running from
the D-V midline and curving up
into the anterior dorsal region
(arrow), and weak N activity is
detected in the ventral part
(arrowhead). (C) Ser (arrows
and arrowheads, green) is
expressed in a domain
corresponding to the lines of
both strong and weak N
activation but occupying
broader areas. (C⬘) Dl (arrows,
red) is upregulated in a line
corresponding to the profile of
strong N activation, and DI
expression can also be seen
weakly in a position
corresponding to the weaker N
activation (arrowhead). (D) Late-third-instar N activity (blue) is detected in the region of the D-V midline and head capsule on both the dorsal
(arrow) and ventral (arrowhead) side. (D⬘) Ser (green) is expressed in a broader region corresponding to N activity. (D⬙) Dl (red) is mainly
expressed behind the MF, but is also expressed at the anterior extreme in both dorsal (arrow) and ventral (arrowhead) regions.
The eye disc portion of the eye-antennal disc contains both the
presumptive retina and head capsule. The presumptive retina
occupies the majority of the tissue extending from the posterior
margin of the disc to an anterior limit defined by the extent of
Eyegone (Eyg) expression (blue in Fig. 6A-B) (Dominguez et al.,
2004). It was found that the Iro+-Iro– interface runs as a straight line
through the eye region, but as it moves into the region of the
presumptive head capsule it curves sharply dorsal (Fig. 6B).
Expression of Dl and Ser was upregulated about this border. Dl
appeared as a stripe in the dorsal cells next to the midline, running
as a straight line through the eye field and curving dorsally with the
interface (red in Fig. 5C⬘). Ser expression was found to be more
complicated: in the region of the eye, it filled the gap vacated by Slp
expression (green in Fig. 6A-B), and as it entered the head capsule
region it bifurcated into two ‘spurs’; the stronger one following the
dorsally curving Iro+-Iro– interface, and the weaker one bending
ventrally (green in Fig. 5C-D and Fig. 6A-B).
Dl and Ser expression in the ventral spur
To determine whether the ventral spur of Ser expression was
associated with N activity, we stained a reporter line for N activity
(N-GV) (Chung and Struhl, 2001). Strong N activity was found in
the region of the Iro+-Iro– interface, both in the midline region of
the eye and in the dorsally curving part in the presumptive head
capsule (arrows in Fig. 5B,D). Additionally, however, N activity
was detected in the region corresponding to the ventral spur of Ser
expression (arrowhead in Fig. 5B,D). This expression is weaker
than its dorsal counterpart, but it was consistently observed. In
addition, a weak upregulation of Dl expression was often detected
at this location (arrowhead in Fig. 5C⬘,D⬙). This ventral spur of N
signaling is distant from and, by inference, independent of the
Iro+-Iro– interface. However, an additional Fng+-Fng– interface
appears to be present in the ventral anterior region of the eye disc
(arrows in Fig. 3B⬘) which corresponds to the position of the
ventral spur of N activity. Thus, this appears to be an Iroindependent Fng+-Fng– interface that regulates N signaling in the
extreme ventral anterior of the eye disc. We note that the mirrlacZ line used in our experiments (mirrF7) is also a w+ enhancer
trap for mirr (McNeill et al., 1997), and in the adult the pigment
fills the dorsal eye stopping at the equator represented by the DV midline (Tomlinson, 2003). Thus, the Iro+-Iro– interface defines
the midline of the eye, and the ventral spur of N activity appears
to play no role in this patterning.
Ser upregulation correlates with downregulation
of slp expression
In second-instar larvae, Ser is expressed about the D-V midline in
both Slp- and Iro-expressing cells. Thus, Ser expression is not
‘generically’ suppressed by these transcription factors, However, Slp
appears to play a role in regulating the subsequent upregulation of
Ser.
In the eye region, the Ser upregulation occurs in the gap left by
the receding slp expression. N expression engenders both these
effects: it upregulates Ser expression (Papayannopoulos et al., 1998)
and downregulates slp expression. In the mid-third larval instar, a
‘peninsula’ of slp expression was seen to project from the main
ventral body of expression and follow the dorsally curving Iro+-Iro–
DEVELOPMENT
Dl and Ser regulation about the Iro+-Iro– interface
Dorsoventral signaling in the fly eye
RESEARCH ARTICLE
665
Fig. 6. (A-A⬙) High levels of Ser
expression (green) correspond with
the loss of slp-lacZ (red) in thirdinstar eye discs. eyg-Gal4/UAS-GFP
expression (blue) demarcates the
eye field (dotted line indicates the
anterior extreme of the eye); Ser
expression anterior to this relates to
patterning of head capsule tissues.
Arrow in A⬘ indicates the
downregulation of slp expression in
the ventral spur corresponding with
high Ser expression. (B-B⬙) Ser
(green) is expressed in cells not
expressing mirr-lacZ (red), except in
the most anterior regions (arrow in
B⬙). (C-D⬙) The roles of N, Slp and
Iro in regulating Ser upregulation in
third-instar discs. (C) Ectopic
expression of slp (red, arrow) in the
region of the gap shows a
downregulation of Ser expression
(green). Note that slp does not
repress Ser completely. (D) Ectopic
expression of mirr in the region of
the gap (red, arrow) shows a
downregulation of Ser expression
(green). Note that the nonautonomous induction of Ser
(arrowhead) is likely to be caused by
the new Fng+-Fng– interface
induced in the ventral cells.
Slp represses Ser expression
As there were two positions in the eye where Ser upregulation
correlated with a downregulation of slp, we wondered whether this
was a causal or a coincidental relationship. Since N activation is
required to repress slp expression, and as Ser is required for that N
activation, we inferred that Ser expression was required for the
downregulation of slp. But was slp downregulation required for the
upregulation of Ser?
slp expressed under a (non-N-inducible) heterologous promoter
in the region of the gap induced a strong reduction in Ser staining
(arrow in Fig. 6C). This was a reduction, not an extinction of Ser
expression, but the effect was clear and reproducible.
Furthermore, expression of mirr also repressed Ser expression
(arrow in Fig. 6D). Thus both Slp and Iro are able to suppress the
upregulation of Ser.
DISCUSSION
We report here that Slp and Iro are mutually repressive transcription
factors expressed in the respective ventral and dorsal domains of the
developing eye. In the second instar the two directly abut at the D-
V midline (Fig. 1E), but N signaling directs the local downregulation
of slp, leading to the emergence of a gap between the two expression
domains (white arrowhead in Fig. 1F). N signaling also instructs the
upregulation of its ligands: Dl expression is increased in dorsal cells
next to the midline, and Ser is expressed in a wider ventral domain
that corresponds to the gap left by the receding slp expression. The
removal of Slp (the formation of the gap) appears necessary for the
effective upregulation of Ser. Below we discuss these results and
address related issues.
slp mutant clones
slp clones in the dorsal region of the developing eye appear normal
and healthy which suggests that there is nothing intrinsically wrong
with slp mutant cells. Yet, when induced in the ventral (Slpexpressing) domain, the clones are sub-viable (white arrowheads in
Fig. 1H-I). Furthermore, the cells that survive are likely to carry
perduring gene function, so we suspect that fully mutant ventral slp
cells do not survive (arrow in Fig. 1G-H). Neither the use of the
Minute technique, nor the presence of the apoptotic inhibitor P35,
affected the survival of the clones. These results suggest that the slp
mutant cells do not suffer a simple growth disadvantage with respect
to their Slp-expressing neighbors, nor are they removed by
apoptosis. Surviving clones (white arrowheads in Fig. 1H) show
smooth borders, which suggests that they are ‘immiscible’ with their
Slp+ neighbors, and that some adhesion mechanism may be
DEVELOPMENT
interface (on its anterior side) (red in Fig. 6A). The ventral spur of
Ser upregulation cut through this peninsula, and here a clear and
correlating downregulation of slp expression was observed (arrow
in Fig. 6A⬘).
RESEARCH ARTICLE
responsible for the removal of the cells. However, we did not observe
extrusion of mutant tissue from the epithelium that would be
expected if ‘immiscibility’ was responsible for the loss of the mutant
cells.
The relationship between Slp and Iro
Slp and Iro are mutually repressive transcription factors: when one
is expressed in the domain of the other, it suppresses the other’s
expression. In dorsal iro mutant clones, slp is transcriptionally
activated (Fig. 2B), indicating that Iro acts to prevent dorsal
expression of slp. The reciprocal experiment of examining iro
transcription in ventral slp clones was confounded by our inability
to generate effectively null slp clones. However, ventral slp clones
induced at all stages throughout larval development showed no
evidence of mirr-lacZ expression (not shown). Furthermore, we note
that the gap is made from ventral cells that turned off slp expression,
and that iro expression does not subsequently turn on in those cells.
We can view the relationship between Slp and Iro in one of two
ways. First, both proteins function in their respective domains to
suppress the expression of the other, and technical problems have
prevented us from detecting the ventral suppression of iro by Slp. It
this were the case, then both would be likely to function to ensure
the emergence of a sharp interface between the two domains, in the
manner described in the Introduction for vertebrate neural tube cell
fates. The second possibility is that Iro represses dorsal slp
expression, but Slp does not repress ventral iro expression.
In this second model, what would be the function of Slp vis-à-vis
Iro? Since Iro suppresses slp (Fig. 2B-C) then this suggests that the
‘ground state’ of the eye tissue is to express slp, and that it is
excluded from dorsal expression by Iro. Wingless (Wg) is required
for Iro expression (Heberlein et al., 1998), and in the embryonic eye
disc primordium slp is expressed in the majority of the eye
primordium, being excluded from the dorsal extreme (not shown).
wg is expressed in the dorsal region (data not shown) and we
speculate that Wg promotes dorsal Iro expression which suppresses
the dorsal expression of Slp. Ventral Slp may then function to repress
any ventral iro expression engendered by diffusing Wg signaling.
The mutual repression of the two transcription factors would then
lead to cells adopting either the Slp–/Iro+ or Slp+/Iro– state.
Immiscibility effects between the cells would then lead to the
formation of the long straight midline border, and in this regard we
note that the surviving slp mutant clones round up when surrounded
by Slp+ cells (arrowheads in Fig. 1H).
Slp and the upregulation of Ser
We have described (in two different positions in the eye disc) that
loss of slp expression correlates with the upregulation of Ser. One
position at which this occurs is the gap that forms next to the Iro+Iro– interface. The other is in the ventral region of the presumptive
head capsule ahead of the eye field; here a spur of Ser expression
shows a corresponding loss/reduction in slp expression (arrow in
Fig. 6A⬘). Not only does the reduction of slp expression correlate
with Ser upregulation, but also restoration of Slp to the region of the
gap prevents the high levels of Ser normally found there (Fig. 6C).
Thus, reduction in slp levels appears necessary for the increase in
Ser levels. We note that slp transcription is monitored here using slplacZ, and as lacZ has significant perdurance, the gap is probably
larger than that indicated, and any overlap between Ser and slp is
likely to be less than that suggested by the images.
In wing D-V patterning there is no known ‘equivalent’ of Slp –
that is, a transcription factor that initially abuts the D-V interface
but is then pushed back by the action of N signaling. Thus, this
Development 134 (4)
role of Slp appears specific to the eye, and this raises the question
of its function. One explanation is that it acts as a brake on the N
signaling. A major difference between the eye and wing is that a
wave of differentiation sweeps the eye, and the midline N
signaling is required specifically ahead of the wave front (the
MF). A delicate balance may be required between the amount of
N signaling ahead of the MF, and the speed with which the MF
progresses across the disc. Slp may provide that control. As a
repressor of Ser upregulation, Slp could act as a delay mechanism
– slowing the increase in Ser, and thereby slowing the increase in
N activation.
We thank Richard Mann for comments on the manuscript; Ken Irvine, Maria
Leptin, Richard Mann, Helen McNeill, Gary Struhl, Leslie Vosshall, Myriam
Zecca, The Developmental Studies Hybridoma Bank and the Bloomington
Stock Center for materials; and Natalya Katanayeva for the germline
transformation. A.S. personally thanks Yoshinori Tomoyasu for helpful
discussion. This work was funded by grant NIH R01EY012536 to A.T.
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Dorsoventral signaling in the fly eye