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First published online 4 October 2006
doi: 10.1242/dev.02593
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Research Report |
1 CABD-Andalusian Centre for Developmental Biology, UPO-CSIC, Sevilla 41013,
Spain.
2 PDBEB, University of Coimbra, Portugal.
3 IBMC, Porto 4150-180, Portugal.
* Author for correspondence (e-mail: fcasfer{at}upo.es)
Accepted 24 August 2006
SUMMARY
Although many of the factors responsible for conferring identity to the eye field in Drosophila have been identified, much less is known about how the expression of the retinal `trigger', the signaling molecule Hedgehog, is controlled. Here, we show that the co-expression of the conserved odd-skipped family genes at the posterior margin of the eye field is required to activate hedgehog expression and thereby the onset of retinogenesis. The fly Wnt1 homologue wingless represses the odd-skipped genes drm and odd along the anterior margin and, in this manner, spatially restricts the extent of retinal differentiation within the eye field.
Key words: Retinal differentiation, Eye, Drosophila, odd genes, Hedgehog, wingless
INTRODUCTION
In Drosophila, the eye primordium is specified as a subdomain of
the Pax6-expressing cells in the center of the eye disc, by the
co-expression of a set of retinal determination genes
(Bonini et al., 1993
;
Cheyette et al., 1994;
Dominguez and Casares, 2005;
Halder et al., 1998;
Mardon et al., 1994;
Pappu and Mardon, 2004). Then,
retinogenesis is triggered by the hedgehog (hh) and the
hh target decapentaplegic (Dpp/Bmp4) signals that are
produced by the surrounding posterior margin cells
(Fig. 1A), at the so-called
`firing point' (Treisman and Heberlein,
1998). These margin cells abut the eye primordium and give rise to
part of the adult head capsule surrounding the eye
(Haynie and Bryant, 1986).
Once initiated, retinal differentiation propagates in a posterior-to-anterior
wave (Fig. 1B,C), with the
differentiation wavefront marked by an epithelial indentation: the
morphogenetic furrow (MF) (Treisman and
Heberlein, 1998). The gene(s) responsible for this specialization
of the posterior margin are unknown.
MATERIALS AND METHODS
Drosophila strains
odd5, drm6, bowl1,
wg1-16 (wgCX3),
oddrK111 (oddZ), hhP30 (hhZ),
dppBS3.0 (dppZ), P{en1}wgen11 (wgZ),
P{GAL4}hhGal4 (hh-GAL4) are described in FlyBase.
Df(2L)drmP2 (Green et al.,
2002; Hao et al.,
2003) deletes from tim to odd, and uncovers
30 predicted genes, including drm, sob and odd. UAS
strains were UAS-odd(A) and UAS-sob(6)
(Hao et al., 2003),
UAS-bowl(1.1) (de Celis Ibeas and
Bray, 2003), UAS-drm (on the III) and UAS-lines
(Green et al., 2002; Hatini et
al., 2000), and UAS-Src-GFP
(Kaltschmidt et al., 2000).
odd-GAL4 faithfully reproduces odd expression (a gift from
G. Morata and M. Calleja, CMB, Spain). drm6 was recombined
onto a FRT40A chromosome.
Loss-of-function clones:
odd5, drm6 and bowl1
mitotic clones were induced between 24 and 48 hours after egg laying (AEL) by
a 45 minute 37°C heat-shock in larvae from the crosses of
odd* FRT 40A/balancer males to yw hsFLP
122; Ubi-GFP FRT40A females (odd* represents each
of the alleles used). DfdrmP2 cells do not survive unless given a
growth advantage, for which we used the `Minute technique'
(Morata and Ripoll, 1975).
Clones were induced between 24 and 72 hours AEL by a 20 minute 37°C
heat-shock in larvae from the crosses of odd*
FRT40A males to yw, hsFLP122; M armZ FRT40A
females. In some experiments, we used yw ey-FLP as flipase source
(Newsome et al., 2000) to
maximize the amount of mutant tissue in eye discs. Mutant cells were
identified by the absence of ß-galactosidase (armZ).
Ectopic-expression ('flip-out') clones of odd-family genes and lines
These clones were induced between 24 and 48 hours AEL (L1 stage) in larvae
from the crosses between UAS-odd* (where
odd* means odd, drm, sob or bowl) or
UAS-lines males and y, hsFLP122,
actinP>hsCD2>Gal4 females
(Basler and Struhl, 1994).
Clones were marked negatively by the absence of CD2 (CD2 was induced by a 45
minute 37°C heat-shock, followed by 45 minutes recovery at room
temperature). The hhZ, dppZ or oddZ reporters were
introduced in the genotypes of some experiments. The overexpression of
drm in bowl- cells was achieved using the MARCM
technique (Lee and Luo, 2001).
UAS-drm was balanced over TM6B, Tb, so
drm-expressing larvae were Tb+. Clones were
marked positively by expression of GFP.
Antibodies
We used rabbit anti-ß-gal (Cappel), mouse anti-ß-gal (Sigma),
rabbit anti-GFP (Molecular Probes), mouse anti-CD2 (Serotec), guinea pig
anti-Odd (Kosman et al., 1998)
and mouse ant-Ptc (Nakano et al.,
1989). Rat anti-Elav, mouse anti-Wg (4D4) and mouse anti-Eya are
from the Iowa University Studies Hybridoma Bank. RNA probes for odd, drm,
sob and bowl were as described previously
(Hao et al., 2003).
Phalloidin-FITC was used to mark filamentous actin. Appropriate fluorescent
secondary antibodies were from Molecular Probes. Anti-mouse-HRP (Sigma) was
used for immunoperoxidase staining.
RESULTS AND DISCUSSION
bowl, odd, drm and sob are expressed in the margin-peripodial cells in early eye discs, but their expression patterns differ later on in development
The eye disc is a flat epithelial sac. By early third larval stage (L3),
columnar cells in the bottom (disc proper: Dp) layer are separated by a crease
from the surrounding rim of cuboidal margin cells. Margin cells continue
seamlessly into the upper (peripodial; Pe) layer of squamous cells
(Fig. 1C-G). The Dp will
differentiate into the eye, while the margin and Pe will form the head capsule
(Haynie and Bryant, 1986). In
addition, the posterior margin produces retinal-inducing signals
(Treisman and Heberlein,
1998).
By examining gene reporters we found that the zinc-finger gene
odd-skipped (odd) is expressed restricted to the posterior
margin and Pe of L3 eye discs (Fig.
1). As the odd family members drumstick (drm),
brother of odd with entrails limited (bowl) and sister
of odd and bowl (sob) are similarly expressed in leg discs
(de Celis Ibeas and Bray,
2003; Hao et al.,
2003), we examined them in eye discs. In L2, before retinogenesis
has started, odd and drm are transcribed in the posterior
Pe-margin (Fig. 1H,I), and this
continues within the posterior margin after MF initiation
(Fig. 1L,M). bowl is
transcribed in all eye disc Pe-margin cells of L2 discs
(Fig. 1J), but retracts
anteriorly along the margins and Pe after the MF passes
(Fig. 1N). In addition,
bowl is expressed weakly in the Dp anterior to the furrow.
sob expression in L2 and L3 is mostly seen along the lateral disc
margins (Fig. 1K,O). Therefore
drm, odd and bowl are co-expressed at the posterior margin
prior to retinal differentiation initiation.
|
; Green et al.,
2002; Hao et al.,
2003; Hatini et al.,
2005; Johansen et al.,
2003). Bowl is required for all these processes
(Green et al., 2002;
Hao et al., 2003). In embryos,
the product of the gene lines
(Bokor and DiNardo, 1996) binds
to Bowl and represses its activity, while Drm relieves this repression in
drm-expressing cells (Hatini et
al., 2005). As drm/odd/bowl expression coincides along
the posterior margin around the time retinal induction is triggered, we asked
whether they controlled this triggering. First, we removed bowl
function in marked cell clones induced in L1. bowl- clones
spanning the margin, but not those in the DP, cause either a delay in, or the
inhibition of, retinal initiation (Fig.
2A,B) and the autonomous loss of hh-Z expression
(Fig. 2C,E). Correspondingly,
there is a reduction in expression of the hh-target patched
(ptc) (Fig. 2D). These
effects on hh and ptc are not due to the loss of margin
cells, as drm is still expressed in the bowl-
cells (not shown). The requirement of Bowl for hh expression is
margin specific, as other hh-expressing domains within the disc
(Royet and Finkelstein, 1997)
are not affected by the loss of bowl (not shown). As expected from
the bowl-repressing function of lines
(Green et al., 2002;
Hatini et al., 2005), the
overexpression of lines along the margin phenocopies the loss of
bowl (Fig. 2F).
Nevertheless, the overexpression of bowl in other eye disc regions is
not sufficient to induce hh (not shown). This suggests that, in
regions other than the margin, either the levels of lines are too
high to be overcome by bowl or bowl requires other factors
to induce hh, or both.
drm and odd are required for and sufficient to initiate retinogenesis
drm and odd are expressed together along the posterior
disc margin-Pe (Fig. 1), and
drm (at least) is required for Bowl stabilization in leg discs
(Hatini et al., 2005).
Nevertheless, the removal of neither drm
(Fig. 3A) nor odd (not
shown) function alone results in retinal defects. odd and
drm may act redundantly during leg segmentation
(Hao et al., 2003) and this
may also be the case in the eye margin. To test this, we induced clones of
DfdrmP2, a deficiency that deletes drm, sob and
odd, plus other genes (Green et
al., 2002). When DfdrmP2 clones affect the margin, the
adjacent retina fails to differentiate, suggesting that drm and
odd (and perhaps sob, for which no single mutation is
available) act redundantly to promote bowl activity at the margin
(Fig. 3B,C) (although we cannot
exclude that other genes uncovered by this deficiency also contribute to the
phenotype). To test the function of each of these genes, we expressed drm,
odd and sob in cell clones elsewhere in the eye disc. Only the
overexpression of drm or odd induced ectopic retinogenesis
(Fig. 3D and not shown), and
this was restricted to the region immediately anterior to the MF, which is
already eye committed. Interestingly, bowl is also expressed in this
region of L3 discs (Fig. 3E).
The retina-inducing ability of drm requires bowl, because
retinogenesis is no longer induced in drm-expressing clones that
simultaneously lack bowl function
(Fig. 3F). Therefore, it seems
that in the eye, drm (and very likely also odd) also
promotes bowl function.
|
|
) or
activation of its pathway (Chanut and
Heberlein, 1995; Dominguez and
Hafen, 1997; Ma and Moses,
1995; Pan and Rubin,
1995; Strutt and Mlodzik,
1995; Wehrli and Tomlinson,
1995) anterior to the furrow is sufficient to generate ectopic
retinal differentiation. As (1) bowl is required for hh
expression at the margin, (2) this hh expression is largely
coincident with that of odd and drm
(Fig. 3G), and (3) drm
(and possibly odd) functionally interacts with bowl, we
checked whether drm- and odd-expressing clones induced the
expression of hh. In both types of clones hh expression is
turned on autonomously, as detected with hh-Z (shown for drm
in Fig. 3H), which would thus
be responsible for the ectopic retinogenesis observed. That the normal
drm/odd/bowl-expressing margin does not
differentiate as eye could be explained if margin cells lack certain eye
primordium-specific factors.
|
;
Treisman and Rubin, 1995).
drm/odd are complementary to wg (monitored by wgZ)
during early L3, when retinal differentiation is about to start, and also
during later stages (Fig.
4A,C,E). In addition, when wg expression is reduced
during larval life in wgCX3 mutants, drm transcription is
extended all the way anteriorly (Fig.
4B,D). This extension precedes and prefigures the ectopic retinal
differentiation that, in these mutants, occurs along the dorsal margin
(Fig. 4B,D,F). Therefore,
wg could repress anterior retinal differentiation by blocking the
expression of odd genes in the anterior disc margin, in addition to its known
role in repressing dpp expression and signaling
(Hazelett et al., 1998;
Treisman and Rubin, 1995).
Interestingly, the onset of retinogenesis in L3 is delayed relative to the
initiation of the expression of drm/odd (this work) and hh
(Cavodeassi et al., 1999;
Cho et al., 2000) in L1-2.
This delay can be explained in three, not mutually exclusive, ways. First, the
relevant margin factors (i.e. drm/odd, hh) might be in place early,
but the eye primordium might become competent to respond to them later. In
fact, wg expression domain has to retract anteriorly as the eye disc
grows, under Notch signaling influence, to allow the expression of
eye-competence factors (Kenyon et al.,
2003). Second, building up a concentration of margin factors
sufficient to trigger retinogenesis might require some time. In fact, the
activity of the Notch pathway along the prospective dorsoventral
border is required to reinforce hh transcription at the firing point
(Cavodeassi et al., 1999).
Third, other limiting factors might exist whose activity becomes available
only during L3. Such a factor might be the EGF receptor pathway, which is
involved in the triggering and reincarnation of the furrow along the margins
during L3 (Kumar and Moses,
2001).
In addition to hh, other genes are required for retinal
triggering, including dpp (Burke
and Basler, 1996; Pignoni and
Zipursky, 1997; Wiersdorff et
al., 1996), eyes absent (eya)
(Bonini et al., 1993) and the
target of eya dachshund (dac)
(Mardon et al., 1994;
Pignoni et al., 1997). These
genes are expressed in both the posterior region of the eye primordium and the
posterior margin. In addition to their role in eye specification, they might
also specify the margin. Although the regulatory relationships between
hh and dpp, or dpp and eya are obscured by
cross-regulatory interactions (Borod and
Heberlein, 1998; Chen et al.,
1999; Curtiss and Mlodzik,
2000; Hazelett et al.,
1998; Pignoni and Zipursky,
1997), recent functional data indicate that dpp and
eya are functionally downstream of hh
(Pappu et al., 2003). The
possibility that the odd genes control the expression or function of
dpp and eya at the margin remains to be tested.
ACKNOWLEDGMENTS
We are grateful to A. Casali, S. Bray, M. Calleja, I. Guerrero, V. Hatini, G. Morata, C. Rauskolb, J Reinitz and I. Rodríguez for reagents, and to J. L. Gomez-Skarmeta, F. Pichaud and C. Rauskolb and members of the laboratory for comments. This work has been funded through grants BMC2003-06248 (Ministerio de Educación y Ciencia, Spain) and POCTI/BIA-BCM/56043/2004 [Fundação para a Ciência e a Tecnologia (FCT), Portugal], which are co-funded by FEDER, to F.C. C.B-P. and J.B. are funded by FCT.
REFERENCES
Basler, K. and Struhl, G. (1994). Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368,208 -214.[CrossRef][Medline]
Bokor, P. and DiNardo, S. (1996). The roles of hedgehog, wingless and lines in patterning the dorsal epidermis in Drosophila. Development 122,1083 -1092.[Abstract]
Bonini, N. M., Leiserson, W. M. and Benzer, S. (1993). The eyes absent gene: genetic control of cell survival and differentiation in the developing Drosophila eye. Cell 72,379 -395.[CrossRef][Medline]
Borod, E. R. and Heberlein, U. (1998). Mutual regulation of decapentaplegic and hedgehog during the initiation of differentiation in the Drosophila retina. Dev. Biol. 197,187 -197.[CrossRef][Medline]
Burke, R. and Basler, K. (1996). Dpp receptors are autonomously required for cell proliferation in the entire developing Drosophila wing. Development 122,2261 -2269.[Abstract]
Cavodeassi, F., Diez Del Corral, R., Campuzano, S. and Dominguez, M. (1999). Compartments and organising boundaries in the Drosophila eye: the role of the homeodomain Iroquois proteins. Development 126,4933 -4942.[Abstract]
Chanut, F. and Heberlein, U. (1995). Role of the morphogenetic furrow in establishing polarity in the Drosophila eye. Development 121,4085 -4094.[Abstract]
Chen, R., Halder, G., Zhang, Z. and Mardon, G. (1999). Signaling by the TGF-beta homolog decapentaplegic functions reiteratively within the network of genes controlling retinal cell fate determination in Drosophila. Development 126,935 -943.[Abstract]
Cheyette, B. N., Green, P. J., Martin, K., Garren, H., Hartenstein, V. and Zipursky, S. L. (1994). The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron 12,977 -996.[CrossRef][Medline]
Cho, K. O., Chern, J., Izaddoost, S. and Choi, K. W. (2000). Novel signaling from the peripodial membrane is essential for eye disc patterning in Drosophila. Cell 103,331 -342.[CrossRef][Medline]
Curtiss, J. and Mlodzik, M. (2000). Morphogenetic furrow initiation and progression during eye development in Drosophila: the roles of decapentaplegic, hedgehog and eyes absent. Development 127,1325 -1336.[Abstract]
de Celis Ibeas, J. M. and Bray, S. J. (2003).
Bowl is required downstream of Notch for elaboration of distal limb
patterning. Development
130,5943
-5952.
Dominguez, M. and Hafen, E. (1997). Hedgehog
directly controls initiation and propagation of retinal differentiation in the
Drosophila eye. Genes Dev.
11,3254
-3264.
Dominguez, M. and Casares, F. (2005). Organ specification-growth control connection: new in-sights from the Drosophila eye-antennal disc. Dev. Dyn. 232,673 -684.[CrossRef][Medline]
Green, R. B., Hatini, V., Johansen, K. A., Liu, X. J. and
Lengyel, J. A. (2002). Drumstick is a zinc finger protein
that antagonizes Lines to control patterning and morphogenesis of the
Drosophila hindgut. Development
129,3645
-3656.
Halder, G., Callaerts, P., Flister, S., Walldorf, U., Kloter, U. and Gehring, W. J. (1998). Eyeless initiates the expression of both sine oculis and eyes absent during Drosophila compound eye development. Development 125,2181 -2191.[Abstract]
Hao, I., Green, R. B., Dunaevsky, O., Lengyel, J. A. and Rauskolb, C. (2003). The odd-skipped family of zinc finger genes promotes Drosophila leg segmentation. Dev. Biol. 263,282 -295.[CrossRef][Medline]
Hatini, V., Green, R. B., Lengyel, J. A., Bray, S. J. and
Dinardo, S. (2005). The Drumstick/Lines/Bowl regulatory
pathway links antagonistic Hedgehog and Wingless signaling inputs to epidermal
cell differentiation. Genes Dev.
19,709
-718.
Haynie, J. L. and Bryant, P. J. (1986). Development of the eye-antenna imaginal disc and morphogenesis of the adult head in Drosophila melanogaster. J. Exp. Zool. 237,293 -308.[CrossRef][Medline]
Hazelett, D. J., Bourouis, M., Walldorf, U. and Treisman, J. E. (1998). decapentaplegic and wingless are regulated by eyes absent and eyegone and interact to direct the pattern of retinal differentiation in the eye disc. Development 125,3741 -3751.[Abstract]
Heberlein, U., Singh, C. M., Luk, A. Y. and Donohoe, T. J. (1995). Growth and differentiation in the Drosophila eye coordinated by hedgehog. Nature 373,709 -711.[CrossRef][Medline]
Johansen, K. A., Green, R. B., Iwaki, D. D., Hernandez, J. B. and Lengyel, J. A. (2003). The Drm-Bowl-Lin relief-of-repression hierarchy controls fore- and hindgut patterning and morphogenesis. Mech. Dev. 120,1139 -1151.[CrossRef][Medline]
Kaltschmidt, J. A., Davidson, C. M., Brown, N. H. and Brand, A. H. (2000). Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system. Nat. Cell Biol. 2,7 -12.[CrossRef][Medline]
Kenyon, K. L., Ranade, S. S., Curtiss, J., Mlodzik, M. and Pignoni, F. (2003). Coordinating proliferation and tissue specification to promote regional identity in the Drosophila head. Dev. Cell 5,403 -414.[CrossRef][Medline]
Kosman, D., Small, S. and Reinitz, J. (1998). Rapid preparation of a panel of polyclonal antibodies to Drosophila segmentation proteins. Dev. Genes Evol. 208,290 -294.[CrossRef][Medline]
Kumar, J. P. and Moses, K. (2001). The EGF receptor and notch signaling pathways control the initiation of the morphogenetic furrow during Drosophila eye development. Development 128,2689 -2697.
Lee, T. and Luo, L. (2001). Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci. 24,251 -254.[CrossRef][Medline]
Ma, C. and Moses, K. (1995). Wingless and patched are negative regulators of the morphogenetic furrow and can affect tissue polarity in the developing Drosophila compound eye. Development 121,2279 -2289.[Abstract]
Mardon, G., Solomon, N. M. and Rubin, G. M. (1994). dachshund encodes a nuclear protein required for normal eye and leg development in Drosophila. Development 120,3473 -3486.[Abstract]
Morata, G. and Ripoll, P. (1975). Minutes: mutants of drosophila autonomously affecting cell division rate. Dev. Biol. 42,211 -221.[CrossRef][Medline]
Nakano, Y., Guerrero, I., Hidalgo, A., Taylor, A., Whittle, J. R. and Ingham, P. W. (1989). A protein with several possible membrane-spanning domains encoded by the Drosophila segment polarity gene patched. Nature 341,508 -513.[CrossRef][Medline]
Newsome, T. P., Asling, B. and Dickson, B. J. (2000). Analysis of Drosophila photoreceptor axon guidance in eye-specific mosaics. Development 127,851 -860.[Abstract]
Pan, D. and Rubin, G. M. (1995). cAMP-dependent protein kinase and hedgehog act antagonistically in regulating decapentaplegic transcription in Drosophila imaginal discs. Cell 80,543 -552.[CrossRef][Medline]
Pappu, K. S. and Mardon, G. (2004). Genetic control of retinal specification and determination in Drosophila. Int. J. Dev. Biol. 48,913 -924.[CrossRef][Medline]
Pappu, K. S., Chen, R., Middlebrooks, B. W., Woo, C., Heberlein,
U. and Mardon, G. (2003). Mechanism of hedgehog signaling
during Drosophila eye development. Development
130,3053
-3062.
Pignoni, F. and Zipursky, S. L. (1997). Induction of Drosophila eye development by decapentaplegic. Development 124,271 -278.[Abstract]
Pignoni, F., Hu, B., Zavitz, K. H., Xiao, J., Garrity, P. A. and Zipursky, S. L. (1997). The eye-specification proteins So and Eya form a complex and regulate multiple steps in Drosophila eye development. Cell 91,881 -891.[CrossRef][Medline]
Royet, J. and Finkelstein, R. (1997). Establishing primordia in the Drosophila eye-antennal imaginal disc: the roles of decapentaplegic, wingless and hedgehog. Development 124,4793 -4800.[Abstract]
Strutt, D. I. and Mlodzik, M. (1995). Ommatidial polarity in the Drosophila eye is determined by the direction of furrow progression and local interactions. Development 121,4247 -4256.[Abstract]
Treisman, J. E. and Rubin, G. M. (1995). wingless inhibits morphogenetic furrow movement in the Drosophila eye disc. Development 121,3519 -3527.[Abstract]
Treisman, J. E. and Heberlein, U. (1998). Eye development in Drosophila: formation of the eye field and control of differentiation. Curr. Top. Dev. Biol. 39,119 -158.[Medline]
Wehrli, M. and Tomlinson, A. (1995). Epithelial planar polarity in the developing Drosophila eye. Development 121,2451 -2459.[Abstract]
Wiersdorff, V., Lecuit, T., Cohen, S. M. and Mlodzik, M. (1996). Mad acts downstream of Dpp receptors, revealing a differential requirement for dpp signaling in initiation and propagation of morphogenesis in the Drosophila eye. Development 122,2153 -2162.[Abstract]
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