1 Section of Cell and Developmental Biology, University of California, San
Diego, 9500 Gilman Drive, La Jolla, CA 92093-0349, USA
2 University of California, San Francisco, Third and Parnassus, San Francisco,
CA 94143-0448, USA
* Author for correspondence (e-mail: bier{at}biomail.ucsd.edu)
Accepted 28 January 2004
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SUMMARY |
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Key words: optomotor-blind, omb, brinker, brk, abrupt, ab, L5 vein, Wing disc, Drosophila, Patterning, Morphogenesis
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Introduction |
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The Bone Morphogenetic Protein (BMP)-related ligand Dpp functions as a
morphogen during several stages of Drosophila development, including
patterning the dorsal/ventral (DV) axis of the embryo
(Bier, 1997;
Rusch and Levine, 1996
) and
establishing the anterior/posterior (AP) axis of the wing disc (reviewed by
Affolter et al., 2001
;
Klein, 2001
;
Lawrence and Struhl, 1996
;
Strigini and Cohen, 1999
). In
the wing disc, Dpp is produced in a stripe just anterior to the AP border, and
diffuses in both anterior and posterior directions to form a concentration
gradient and a corresponding BMP activity gradient
(Entchev et al., 2000
;
Fujise et al., 2003
;
Klein, 2001
;
Lawrence and Struhl, 1996
;
Strigini and Cohen, 1999
;
Teleman and Cohen, 2000
). This
BMP activity gradient, which is established by the synergistic action of the
ligands Dpp and Glass Bottom Boat (Gbb)
(Haerry et al., 1998
;
Wharton et al., 1999
),
functions in a dosage-sensitive fashion to control the nested expression of a
series of BMP target genes. The BMP target genes spalt-major
(salm) and spalt-related (salr) (these related and
neighboring genes will be referred to as sal hereafter),
optomotor-blind (omb; bifid, bi FlyBase),
and vestigial (vg) are expressed in progressively broader
domains due to their increasing sensitivity to BMP signaling
(Kirkpatrick et al., 2001
;
Lecuit et al., 1996
;
Nellen et al., 1996
).
A crucial Dpp target gene is brinker (brk), which is
repressed in a graded fashion by Dpp signaling in the central region of the
wing disc (Marty et al., 2000;
Muller et al., 2003
;
Torres-Vazquez et al., 2000
).
Brk encodes a transcriptional repressor
(Campbell and Tomlinson, 1999
;
Jazwinska et al., 1999a
;
Minami et al., 1999
) that acts
in a dosage-dependent manner to establish the centrally nested expression of
the transcription factors encoded by omb, sal and vg
(Jazwinska et al., 1999b
;
Sivasankaran, 2000; Kirkpatrick et al.,
2001
; Muller et al.,
2003
). Thus, the opposing and complementary activities of Dpp and
Brk along the AP axis of the wing disc lead to differential activation of
target genes such that the more responsive a gene is to BMP signaling and the
less sensitive it is to repression by Brk, the broader its expression domain
will be.
Although much is understood regarding the formation of the Dpp gradient and
how the resulting graded activation of BMP signaling elicits different
patterns of gene expression, little is known about how these target genes
direct differentiation of defined tissues in specific locations. A mechanism
that links broad patterns of gene expression to specification of particular
cell types is the creation of sharp borders between different domains. As
described above, graded BMP signaling subdivides the wing disc into nested
domains expressing the target genes sal, omb and vg. These
expression domains create boundaries that then can act as local organizers
along the AP axis (Lawrence and Struhl,
1996) to induce formation of specific morphological structures
such as longitudinal wing veins (Bier,
2000
; Sturtevant et al.,
1997
; Sturtevant and Bier,
1995
).
A well-studied example of vein induction at a boundary is formation of the
L2 primordium along the anterior border of the sal expression domain.
Cells expressing high levels of sal induce expression of
knirps (kni) and knirps related (knrl;
knirps-like FlyBase) genes in a narrow stripe of neighboring
anterior cells, which express low levels of sal
(de Celis and Barrio, 2000;
Lunde et al., 1998
). Analysis
of an L2 vein-specific enhancer element of the kni locus revealed
that it consists of an activation domain containing functionally important
Scalloped (Sd)-binding sites, as well as a repressor domain containing
consensus binding sequences for Sal and Brk
(Lunde et al., 2003
). Kni/Knrl
organize development of the L2 primordium by activating expression of the vein
promoting gene rhomboid (rho), as well as by repressing
expression of the intervein gene blistered (bs) in the vein
primordial cells (Lunde et al.,
1998
). In addition, because Kni/Knrl expression must be confined
to a narrow stripe to promote vein development
(Lunde et al., 1998
), it may
also control expression of a lateral inhibitory factor that represses vein
development in adjacent intervein cells. Therefore, Kni/Knrl play a key role
in translating positional information at the anterior border of the
sal expression domain into a coherent gene expression program in the
L2 primordium.
The position of the L5 primordium, like that of L2, is determined by a
threshold response to the Dpp gradient
(Sturtevant et al., 1997).
However, the border(s) of gene expression domains responsible for inducing
formation of L5 are unknown. In this study we examine the initiation of L5
development and show that it is dependent on the two abutting Dpp target
genes, omb and brk. The L5 primordium forms within the
omb domain adjacent to cells expressing high levels of brk.
We show that omb is required for responding to a for-export-only
signal produced by brk-expressing cells. This combination of
constraints results in the activation of abrupt (ab), which
plays a key role in organizing gene expression in a sharp line within the
posterior extreme of the omb expression domain.
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Materials and methods |
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Clonal analysis
Homozygous loss-of-function clones were generated by hsFLP-FRT
recombination (Xu and Rubin,
1993). y w brkm68 f36a FRT18a/FM7a
and ombD4 w/FM6 stocks were recombined to
generate the w ombD4 f36a FRT18a/FM0 and w
ombD4 brkm68 f36a FRT18a/FM0 stocks.
Each of these stocks was crossed with w[1118] P{Ubi-GFP(S65T)nls}X
P{neoFRT}18A; MKRS, P{hsFLP}86E/TM6B, Tb[1] and larvae were heat shocked
24-72 hours after egg-laying at 37°C for 1-2 hours. Wing discs were
dissected and analyzed after 24-72 hours, or vials were kept at 25°C until
flies hatched and wings were analyzed. Mutant clones in the wing disc were
detected by lack of GFP expression, and in the adult wing by
f36a phenotype.
Flip-out clones ectopically expressing ab were generated in larvae of the genotype yw hsFLP f36a; ab>f+>GAL4-lacZ/UAS-ab following recombination between FRT elements (>), initiated by heat induction of the HS-FLP recombinase transgene for 30 minutes at 34°C. These clones were marked by gain of lacZ expression in the disc, and by the cell-autonomous f36a trichome phenotype in adult wings. A similar set of crosses was used to generate flip-out clones misexpressing high levels of omb.
Generation of an anti-Abrupt antibody
An Abrupt-GST fusion protein consisting of the 88 C-terminal amino acids of
Ab fused to GST was purified from soluble whole bacterial protein extracts,
using a glutathione column, and injected into rabbits. The antiserum was
partially purified by ammonium-sulfate precipitation (25% cut) and preabsorbed
1:10 against fixed embryos. Titration of this antibody revealed that a final
1:1000 dilution gave a strong signal with low background.
Immunostaining
Immunohistochemical staining was performed using the following antibodies:
Guinea pig anti-Kni (kindly provided by D. Kosman), mouse anti-Delta (kindly
provided by M. Muskavitch), mouse anti-DSRF (kindly provided by M. Affolter),
mouse anti-ß-Gal (Promega), and rabbit ß-Gal (Cappel), as previously
described in (Sturtevant et al.,
1993). Fluorescent detection using secondary Alexa Fluor 488, 555,
594 or 647 conjugated antibodies (Molecular Probes) was visualized using a
Leica scanning confocal microscope.
In situ hybridization to whole-mount larval wing discs
In situ hybridization using digoxigenin-labeled antisense RNA probes was
performed either alone (O'Neill and Bier,
1994) or in combination with antibody labeling, as previously
described (Sturtevant et al.,
1993
).
Mounting fly wings
Wings from adult flies were dissected in ethanol and mounted in 50% Canada
Balsam (Aldrich #28,292-8), 50% methylsalicilate, as described by Ashburner
(Ashburner, 1989).
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Results |
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We also investigated whether restricted expression of ab in small
clones was sufficient to induce vein development. We used the flip-out
misexpression system (Struhl and Basler,
1993) to generate clones of cells ectopically expressing
ab in the wing disc, and found that these cells (identified by Ab or
ß-Gal expression) ectopically expressed the vein marker Dl
(Fig. 1O,P) and downregulated
expression of the intervein marker Bs (Fig.
1Q,R) in a cell-autonomous fashion when located anywhere within
the wing pouch. Adult wings containing small ab-expressing clones
marked with forked also produced ectopic vein material cell
autonomously (Fig. 1S). These
results, in conjunction with those described above, demonstrate that
ab is necessary to control known gene expression in the L5
primordium, and is sufficient to induce vein development when expressed in a
restricted number of cells. These data are consistent with ab acting
in a vein-organizing capacity to direct L5 development.
ab is expressed along the border of omb and brk expression domains
As previously shown, the L2 primordium forms along the anterior boundary of
the sal expression domain, in cells expressing low levels of
sal and facing those expressing high levels of sal
(de Celis and Barrio, 2000;
Lunde et al., 1998
;
Sturtevant et al., 1997
). The
symmetrical disposition of the L2 and L5 veins, and the positioning of both of
these veins by Dpp rather than Hh signaling, suggested that the L5 vein might
form along the posterior border of the sal expression domain in much
the same way that L2 is induced along its anterior border. However, two lines
of evidence indicate that sal is not likely to be directly involved
in determining the position of L5. First, the posterior border of the
sal expression domain is located several cells anterior to the L5
primordium (Sturtevant et al.,
1997
). Second, although salm- clones do
occasionally result in the formation of ectopic posterior veins, they do so
non-autonomously at a distance of several cell diameters from the clone border
(Sturtevant et al., 1997
).
This phenotype is entirely different from the ectopic L2 veins that form at
high penetrance immediately within the borders of anterior
sal- clones, located between the L2 and L3 veins
(Sturtevant et al., 1997
).
Clones of a deficiency removing both salm and the related
salr gene also result in the production of an ectopic vein
(de Celis and Barrio, 2000
),
but this vein forms within the interior of such clones between L4 and L5, in a
position corresponding to a cryptic vein, or paravein, which has a latent
tendency to form along the posterior border of the sal domain
(Sturtevant et al., 1997
).
As the L5 primordium forms approximately four to six cell diameters
posterior to the sal expression domain
(Fig. 2A-C)
(Sturtevant et al., 1997), we
examined the expression of other BMP target genes, omb and
brk, relative to the L5 primordium. The borders of these gene
expression domains are known to form posterior to that of the sal
domain (Campbell and Tomlinson,
1999
; Jazwinska et al.,
1999a
; Lecuit et al.,
1996
; Minami et al.,
1999
; Nellen et al.,
1996
). Previous studies revealed that the domains of cells
expressing high levels of omb and brk
(Campbell, 2002
;
Jazwinska et al., 1999a
) are
largely reciprocal, although these genes are co-expressed at lower levels in
cells along the border. We therefore determined the relative positions of the
border of high level omb/brk expression with respect to vein
primordia marked by Dl (L1, and L3-L5) and Kni (L2). These experiments
revealed that the L5 stripe of Dl expression forms inside and along the
posterior border of the domain expressing high levels of omb, whereas
the anterior border of the omb domain extends well beyond the L2
primordium (Fig. 2D-F). A
complementary pattern was observed in wing discs of brk-lacZ flies
double stained for ß-Gal and Dl, in which the L5 Dl stripe runs outside
and along the border of the high level brk expression domain
(Fig. 2G-I). We obtained
similar results using ab as a marker for the L5 primordium, in which
we found that the stripe of ab-expressing cells lies within the
omb domain (Fig. 2J),
adjacent to high level brk-expressing cells
(Fig. 2K). These expression
studies reveal that omb and brk are expressed in the right
location to play a role in positioning the L5 primordium.
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To eliminate or blur the borders between brk and omb
expressing cells we misexpressed omb or brk with weak or
strong ubiquitous wing drivers, as well as the VgB-GAL4
driver, which activates localized gene expression along the wing margin
(Fig. 4C)
(Williams et al., 1994).
Ubiquitous misexpression of either omb or brk in the wing
using the strong MS1096-GAL4 driver resulted in small wings with a
range of venation phenotypes, in which all or some veins were shifted,
truncated or missing entirely (e.g. Fig.
4A). In these experiments the L2 and L5 veins were particularly
sensitive to the effects of ubiquitous brk or omb
expression, although other veins were also disrupted by high expression levels
of these genes (data not shown). The global effects on wing patterning
associated with strong ubiquitous expression of omb or brk
may result from disrupting more general functions of these primary BMP
response genes in defining regional identities within their broad domains of
expression.
|
Misexpression of omb at modest levels also caused specific venation defects. For example, ectopic expression of omb along the posterior wing margin driven by VgB-GAL4 (Fig. 4C) causes distal truncation of the L5 primordium near its intersection with the margin (Fig. 4E, arrow). This loss of the endogenous L5 primordium may be a consequence of reduced brk expression in these cells since ectopic omb expression in peripheral regions of the wing disc results in downregulation of brk expression (O.C., unpublished). Another consequence of misexpressing omb with the VgB-GAL4 driver is the creation of a new brk/omb border posterior to L5. This border forms between the narrow strip of VgB-GAL4 expressing cells and the posterior edge of the endogenous brk expression domain, as can be observed in brk-lacZ; VgB-GAL4; UAS-GFP wing discs (Fig. 4C, arrow). In a fraction of VgB-GAL4; UAS-omb flies, we observed ectopic veins forming posterior to L5 (Fig. 4E, arrowhead), in addition to the posterior truncation of the endogenous L5 vein. This ectopic vein forms in the expected location of the new brk/omb border created by VgB-GAL4>omb expression. These observations suggest that having a sharp posterior omb/brk boundary is important for L5 formation.
omb is required cell autonomously for L5 development
In order to determine whether the boundary between brk and
omb expression domains was necessary for inducing L5 development, we
generated somatic brk or omb mutant clones or double-mutant
clones lacking both brk and omb function (see Materials and
methods for details). We first examined the requirement for omb by
generating omb- null clones in different regions of the
wing. Such omb- clones did not result in any vein
phenotype when they were located in central regions of the wing
(Fig. 5C, arrowhead), although
these cells normally express high levels of omb in wing discs. This
result indicates that simply having a border between omb expressing
and non-expressing cells is not sufficient to induce vein formation. Moreover,
although omb expression also extends into the L2 primordium
(Fig. 2D-F), omb- clones located in this region of the wing did not
disrupt formation of the L2 vein or expression of the L2 organizer gene
kni in wing discs (Fig.
5A,B). By contrast, omb- clones in posterior
regions of the wing that overlapped part of the L5 vein resulted in vein loss
within the clone (Fig. 5C,
arrow). Consistent with the L5 vein-loss adult phenotype, cells within
omb- clones crossing the L5 primordium in third instar
wing discs failed to express the vein marker Dl
(Fig. 5D), whereas
omb- clones located in central regions of the wing disc
had no effect on Dl expression in the L3 or L4 primordia (data not shown).
These data indicate that omb is required specifically for the
formation of the normal L5 primordium, although a border of omb
expression domain is not sufficient on its own to induce vein formation.
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Anterior brk- clones located several cell diameters away from the endogenous L2 vein also induced ectopic veins running within and along the clone border (Fig. 5G, red arrowhead). In third instar wing discs, comparably located clones ectopically expressed the L2 vein organizing gene kni within the clone (Fig. 5H). However, brk- clones located in the immediate vicinity of the endogenous L2 vein induced ectopic veins along the outside border of the clone (Fig. 5G, black arrowhead). The potential basis for the different behaviors of anterior brk- clones as a function of distance from the L2 vein is discussed below.
The above analysis of the brk- and
omb- single mutant clones suggests that
brk-expressing cells induce L5 development in adjacent
omb-overexpressing cells. This condition is met when
brk- clones are generated in posterior regions of the
wing, as loss of brk activity in these cells results in de-repression
of omb expression (Sivasankaran
et al., 2000). To test whether omb expression is required
for the induction of ectopic veins within brk- clones, we
generated omb- brk- double mutant clones. In
contrast to brk- single mutant clones, we did not observe
consistent induction of veins running along the edges of omb-
brk- clone borders. In many cases, double-mutant
omb- brk- clones contained no veins at all. In
other cases, patches of vein material were observed that tended to be either
short fragments of vein, which did not follow the clone boundary, or diffuse
random veins meandering within the clone
(Fig. 5I). Consistent with this
adult wing-vein phenotype, posteriorly located double-mutant
omb- brk- clones in third instar wing discs did
not induce expression of the vein marker Dl along clones borders. In
some of these omb- brk- clones, we observed
diffuse expression of Dl or fragments of internal Dl expression
(Fig. 5J), and in other cases
we observed no Dl expression at all (data not shown). One unexpected result
was that although omb is not required for formation of the endogenous
L2 vein, it is essential for formation of ectopic veins observed in anteriorly
located brk- clones. Thus, in contrast to the ectopic
veins which formed along the inside borders of anterior
brk- single clones, similarly positioned
omb- brk- double mutant clones generally did
not form any ectopic veins (Fig.
5K), nor did they induce ectopic Kni expression within the clone
boundary (Fig. 5L). This
finding suggests that the ectopic veins in anterior brk-
clones may not have a simple L2 identity (see Discussion below). Cumulatively,
this clonal analysis reveals that induction of the L5 primordium depends on
two conditions being met: (1) cell-autonomous Omb activity; and (2)
non-autonomous induction by Brk acting across a sharp border with adjacent
omb-expressing cells.
ab acts downstream of brk in L5 development
As ab functions at an early stage in L5 development (i.e. as a
vein organizing gene), we investigated whether Ab was also misexpressed along
the border of brk- clones. We found that, as in the case
of Dl, a ring of ectopic Ab expression circumnavigated the interior border of
brk- clones located in the vicinity of the endogenous L5
primordium (Fig. 5N). By
contrast, no such Ab expression was observed in omb-
brk- double mutant clones (data not shown). These results are
consistent with activation of ab expression being downstream of a
brk-induced signaling event.
We also determined whether ab is required to mediate the formation of ectopic veins observed in brk- clones. We addressed this question by generating brk- clones in an ab1/ab1 mutant background and scoring adult-vein phenotypes in various regions of the wing primordium. This analysis revealed that the frequency of ectopic veins within clones located in the vicinity of L5 was significantly reduced in brk- clones produced in ab1/ab1 versus wild-type backgrounds. Some clones that formed posterior to L5 resembled omb- brk- double mutant clones, in that they either lacked veins entirely or had veins running diffusely within the clone region but not along the boundary (Fig. 5M, blue outlined clone). In larger clones, veins followed the clone border in proximal regions of the wing for a short distance, and then ended as the clone entered the distal regions (Fig. 5M, red arrowhead; compare with Fig. 5E), where the endogenous L5 vein is truncated in ab1/ab1 mutants (Fig. 5M, black arrowhead; see legend for quantification). These results suggest that ab is an essential mediator of brk- and omb-dependent induction of the L5 primordium.
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Discussion |
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Induction of L5 formation along the brk/omb border
In a previous model for establishing the position of the L5 primordium, it
was proposed that sal/salr was the only Dpp target gene responsible
for wing vein patterning, which determined the anterior position of the L5
primordium by repressing expression of IroC genes
(de Celis and Barrio, 2000).
It was also suggested that IroC gene expression was directly dependent on BMP
signaling and that fading of the BMP activity gradient determined the
posterior limit of IroC gene expression
(de Celis and Barrio,
2000
).
In the current study, we examined the role of two other Dpp target genes, which are expressed in domains abutting (brk) or just including (omb) the L5 primordium, in establishing the position of this vein. Our results, suggest an alternative model for how the BMP activity gradient induces formation of the L5 primordium in the posterior compartment of the wing (Fig. 6). According to this model, L5 development is initiated within the posterior region of the wing where brk and omb are expressed in adjacent domains with a sharp border between them. As brk- clones induce vein development within the clone along the border with brk+-neighboring cells, we suggest that brk-expressing cells produce a short-range vein-inductive signal, Y, to which they cannot respond. This signal acts on neighboring omb-expressing cells to initiate vein development. The additional cell-autonomous requirement for Omb activity to respond to this Brk-derived signal suggests that the intracellular effector of the vein inductive signal Y must act in combination with Omb to induce vein formation. Because Brk is a repressor of omb expression, the combined requirement for the short-range Brk-derived vein-inductive signal and Omb activity within responding cells constrains L5 initiation to omb-expressing cells adjacent to brk-expressing cells. In this scheme, Brk plays at least two distinct roles in L5 induction. First, as a repressor of omb, Brk defines the border between the brk and omb expression domains, and, second, brk-expressing cells are the source of a vein-inductive signal required to initiate L5 development within adjacent omb-expressing cells.
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A curious phenotype associated with some brk- clones generated in an ab1/ab1 background is the formation of diffuse wandering veins within the interior of the clone. A similar disorganized ectopic vein phenotype is also observed in a fraction of omb- brk- double mutant clones. This phenotype may reflect the lack of a lateral inhibitory factor (e.g. Dl) produced by ab-expressing cells to suppress vein formation in neighboring cells. The observation that ubiquitous expression of ab suppresses vein formation throughout the wing disc is consistent with this possibility. It is also possible that omb plays a role in promoting intervein development as well as in activating ab expression. Additional analysis will be needed to address this question.
Brk plays a role in positioning the L2 primordium
Previous analysis of L2 initiation lead to a model in which
sal-expressing cells produce a short-range vein-inductive signal (X)
to which they cannot respond (Fig.
6) (Sturtevant et al.,
1997). In response to signal X, neighboring cells outside of the
sal domain express the L2 vein-organizing genes kni and
knrl (Bier, 2000
;
Lunde et al., 1998
). In
addition, analysis of an L2-specific cis-regulatory element of the
kni/knrl locus provided indirect evidence for negative
regulation by a repressor, possibly Brk, expressed in peripheral/lateral
regions of the wing disc (Lunde et al.,
2003
).
In the current study, we find that anterior brk- clones
result in two different phenotypes, depending on their distance from the L2
primordium. First, as suspected from analysis of the L2-enhancer element, Brk
acts in a cell-autonomous fashion to repress kni/knrl
expression. This effect of Brk is observed in clones located several cell
diameters anterior to the L2 primordium. The cell-autonomous induction of
veins within the borders of these brk- clones can be
explained by a mechanism similar to that operating within the posterior
compartment, where brk-expressing cells induce vein development in
adjacent cells. In such clones, loss-of-brk function does not result
in significant levels of ectopic sal expression
(Campbell and Tomlinson, 1999)
(O.C., unpublished). The absence of a vein outside of these clones could
result from a combination of three effects. First, the low levels of
sal in such clones is not likely to be sufficient to activate
expression of appreciable levels of signal X. Second, Brk levels outside of
the clones are higher than in the L2 region, which presumably represses
kni expression effectively in cells surrounding the
brk- clones. Finally, there is evidence that low levels of
salr and/or salm are required for L2 development
(de Celis and Barrio, 2000
),
and detectable endogenous expression of sal extends only a short
distance beyond the L2 primordium.
The second phenotype associated with brk- clones, which
is restricted to clones located immediately anterior to L2, is a cell
non-autonomous effect in which short segments of vein form along the clone
border just outside of the clone. This non-autonomous effect of
brk- clones located at branch points with L2 may be
explained by the de-repression of Sal within such clones. As sal
expression also requires a positive input from the BMP pathway, relieving
repression by Brk induces high levels of Sal for only a short distance
anterior of the L2 primordium (Campbell and
Tomlinson, 1999) (O.C., unpublished). These
sal-expressing cells should produce the L2 inductive signal X, which
acts in a cell non-autonomous fashion, as proposed in the model for L2
formation (Fig. 6).
An interesting question regarding veins forming within more anteriorly
located brk- clones is do they have an L2- or an L5-like
identity? On the one hand, these veins express kni, but not Dl,
suggesting that they have an L2-like identity. On the other hand, the ectopic
veins induced anteriorly by brk- clones require
omb function, as do L5-like veins generated in the posterior
compartment of the wing. This latter observation suggests that the
brk- border in anterior regions acts as it does in
posterior regions of the wing disc, but that its effect may be mediated by the
L2 organizing kni/knrl locus rather than the L5 organizing
gene ab. This hypothesis might provide an explanation for why ectopic
veins that form in various mutant backgrounds tend to form along a line
running between the L2 vein and the margin (which we refer to as the P2
paravein) (Sturtevant et al.,
1997). This sub-threshold vein promoting position may be defined
by the anterior border of brk and omb expression. Further
analysis of the identity of these ectopic veins will be required to resolve
this question.
Similarities and differences between induction of the L2 and L5 vein primordia
As the L2 and L5 veins form at similar lateral positions within the
anterior and posterior compartments of the wing, respectively, it is
informative to compare the mechanisms by which positional information is
converted into vein initiation programs in these two cases. The positions of
these two veins are determined by precise dosage-sensitive responses to BMP
signaling emanating from the center of the wing, which are mediated by the
borders of the broadly expressed, Dpp signaling target genes sal and
omb. Brk also plays a role in initiating both L2 and L5 development.
In the posterior compartment, Brk leads to the production of a hypothetical
vein-promoting signal Y, which has a similar function and range as the
putative L2 vein-inducing signal X, produced by sal-expressing cells.
It is not clear whether the signals X and Y are the same or different;
however, an important difference between L2 and L5 initiation is that only L5
has an additional requirement for omb function. This dual requirement
for omb function within the L5 vein primordium and a short-range
inductive signal in neighboring brk-expressing cells provides a
stringent constraint on where the L5 primordium forms. Brk may also directly
repress expression of the vein-organizer gene ab in cells posterior
to the L5 primordium, in analogy to its proposed role as a repressor of
kni/knrl anterior to L2. One possible rationale for
induction of the L5 vein depending on inputs from both omb and
brk is that these genes are expressed in partially overlapping
patterns and neither pattern may carry sufficiently detailed information to
specify the position of the L5 primordium alone. Although the omb and
brk expression levels fall off relatively steeply (i.e. over a
distance of six to eight cells), these borders are not as sharp as the
anterior sal border (two to three cells wide), which alone is
sufficient to induce the L2 primordium.
A final similarity between the initiation of L2 and L5 formation is that
induction of both veins is mediated by a vein-organizing gene that regulates
vein and intervein gene expression in the vein primordium. Although
kni and ab are members of different subfamilies of Zn-finger
transcription factors, they are both expressed in a narrow stripe of cells
along their respective inductive borders, and ubiquitous misexpression of
either gene [see Fig. 1 for
ab, and see Lunde et al.
(Lunde et al., 1998) for
kni] results in elimination of vein pattern in the wing disc. Thus,
the L2 and L5 veins are induced by remarkably similar mechanisms and
principles of organization. Further comparison of the mechanisms of these
developmental programs should provide insights into the degree to which
general and specific vein processes define the L2 versus the L5 vein
identity.
Boundaries translate graded positional information into sharp linear responses
Induction of Drosophila wing veins at borders between adjacent
gene expression domains provides a simple model system for studying how
information provided by morphogen gradients is converted into the stereotyped
pattern of wing vein morphogenesis. Each of the four major longitudinal veins
(L2-L5) is induced by a for-export-only mechanism in which cells in one region
of the wing produce a diffusible signal to which they cannot respond. In the
case of L3 and L4, an EGF-related signal (Vein) is produced between these
veins in the central organizer where expression of the EGF receptor is locally
downregulated (Crozatier et al.,
2002; Mohler et al.,
2000
; Vervoort et al.,
1999
). With respect to L2, response to the vein-inductive signal X
is repressed in Sal-expressing cells that produce the hypothetical signal X
(Lunde et al., 1998
;
Sturtevant et al., 1997
).
Finally, the L5 vein-inductive signal produced by brk-expressing
cells depends on omb, the expression of which is repressed by
Brk.
For-export-only mechanisms also underlie the induction of boundary cell
fates in many other developmental settings. In the well-studied
Drosophila wing, the earliest and most rigorously defined boundaries
are the AP and DV borders, which are determined by Hh and Notch signaling,
respectively. These compartmental borders define domains of non-intermixing
groups of cells, and function as organizing centers by activating expression
of the long-range morphogens Dpp and Wingless (Wg), respectively (reviewed by
Sanson, 2001). In both cases,
cells in one compartment produce a signal to which they cannot respond. This
signal is constrained to act only on neighboring cells in the adjacent
compartment. Other well-studied examples of for-export-only signaling include:
induction of the mesectoderm in blastoderm stage Drosophila embryos
by a likely cell-tethered Notch ligand expressed in the mesoderm
(Cowden and Levine, 2002
;
Lecourtois and Schweisguth,
1995
; Lunde et al.,
1998
; Morel et al.,
2003
; Morel and Schweisguth,
2000
); induction of parasegmental expression of stripe
via Wg, Hh and Spi signaling in gastrulating Drosophila embryos
(Hatini and DiNardo, 2001
);
induction of mesoderm in Xenopus embryos by factors produced in the
endoderm under the control of VegT (reviewed by
Shivdasani, 2002
); and
formation of the DV border of leaves in plants controlled by the
PHANTASTICA gene (Waites et al.,
1998
). The similar but distinct mechanisms for inducing the L2 and
L5 vein primordia offers a well-defined system for examining these relatively
simple cases in depth. These inductive events take place at the same
developmental stage but within separate compartments of a single imaginal
disc, and should provide general insights into the great variety of mechanisms
that can be co-opted to accomplish for-export-only signaling.
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ACKNOWLEDGMENTS |
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