- Research article
- Open Access
Spatial discontinuity of Optomotor-blind expression in the Drosophila wing imaginal disc disrupts epithelial architecture and promotes cell sorting
© Shen et al; licensee BioMed Central Ltd. 2010
Received: 21 September 2009
Accepted: 23 February 2010
Published: 23 February 2010
Decapentaplegic (Dpp) is one of the best characterized morphogens, required for dorso-ventral patterning of the Drosophila embryo and for anterior-posterior (A/P) patterning of the wing imaginal disc. In the larval wing pouch, the Dpp target gene optomotor-blind (omb) is generally assumed to be expressed in a step function above a certain threshold of Dpp signaling activity.
We show that the transcription factor Omb forms, in fact, a symmetrical gradient on both sides of the A/P compartment boundary. Disruptions of the Omb gradient lead to a re-organization of the epithelial cytoskeleton and to a retraction of cells toward the basal membrane suggesting that the Omb gradient is required for correct epithelial morphology. Moreover, by analysing the shape of omb gain- and loss-of-function clones, we find that Omb promotes cell sorting along the A/P axis in a concentration-dependent manner.
Our findings show that Omb distribution in the wing imaginal disc is described by a gradient rather than a step function. Graded Omb expression is necessary for normal cell morphogenesis and cell affinity and sharp spatial discontinuities must be avoided to allow normal wing development.
The concept of Dpp as morphogen in early wing development owes much to the observation of nested target gene expression domains, initially described for spalt (sal) and omb [1, 2] and subsequently for vestigial (vg) and the vg quadrant enhancer . Dpp spreads from its expression domain along the A/P compartment boundary to receiving cells forming a gradient which directs patterning and growth of the wing pouch [4–6]. Dpp signaling represses the transcriptional repressor gene brinker (brk), which is thereby expressed in a gradient inverse to the Dpp signaling activity . The relationship between target gene expression and Brk level is not simply reciprocal. For instance, high level sal expression in the central wing pouch requires direct Dpp signaling in addition to repression of brk, i. e. the sal expression domain is specified by opposing gradients [8–10]. Moreover, different target genes appear to be repressed by Brk through different mechanisms . Irrespective of the mechanistic details, the nested expression pattern of sal and omb forms the basis of the threshold model of Dpp (or rather Brk) target gene regulation [12, 13].
Apart from setting up gene expression patterns, the Dpp gradient appears to fulfil additional roles. Dpp is required for establishing a density gradient of the apical microtubule web (AMW), a specialization of the columnar wing pouch epithelium [14, 15]. Clonal reduction of Dpp signaling in the disc main epithelium leads to wing size reduction and JNK-dependent cell death [16–19]. Mutant cells cluster into cysts in which apical and baso-lateral contacts to neighbouring wild type cells are disrupted [15, 20, 21]. These changes are not secondary to the activation of the JNK-pathway and apoptosis (which are elicited at the junction of cells strongly differing in Dpp signaling activity ) because they occur when these processes are inhibited. The mutant cysts can survive to the adult stage and differentiate. One recognized feature of cytoskeletal reorganization in tkv clones is the loss of the AMW. Taken together, these findings led to the hypothesis that the Dpp gradient ensures correct cell morphogenesis which is necessary for epithelial integrity [15, 20]. Clones mutant for the Dpp signal transducer Mothers against dpp (Mad) lose the AMW and are extruded from the epithelium. Clone extrusion is suppressed in mad brk double mutant clones, suggesting that this function of Dpp is mediated by Brk-mediated Dpp target genes [15, 20, 21]. As the Dpp target gene omb is required to maintain normal epithelial structure at the A/P boundary [22, 23], omb might be, and here is demonstrated to be, one of the mediators downstream of Dpp signaling in the establishment of epithelial architecture also elsewhere in the wing disc.
The Drosophila wing imaginal disc is subdivided into an anterior and a posterior compartment. In analogy to other systems, the segregation of cells at the A/P boundary is thought to be due to compartmental differences in cell-cell affinity (cell affinity hypothesis [24, 25]), although recent analysis indicates an important role for increased mechanical tension for the maintenance of the A/P boundary . Omb cooperates with Hh signaling to promote cell segregation at the A/P boundary, presumably by regulating the expression of cell affinity molecules .
Omb is essential for wing development and is sufficient to initiate secondary wing morphogenesis when ectopically expressed in the notum anlage of the wing disc . The homologous vertebrate Tbx2 subfamily genes (Tbx2-Tbx5) are also involved in limb development and cause inherited haploinsufficiency syndromes associated with limb defects when mutated in humans (TBX3-TBX5) [29–31]. Tbx2 and Tbx3 are amplified or overexpressed in a wide range of neoplasms. Increased levels of TBX2/3 contribute to cancer progression by suppressing cellular senescence and by promoting invasiveness (reviewed in ). The latter phenomenon may be related to the morphogenetic role of Omb which we describe here.
The nested expression patterns of Sal and Omb are generally taken to support a threshold model of Dpp (or rather Brk) target gene regulation, e.g. [1, 2, 8, 10, 12, 13, 33, 34]. We show that, contrary to prior interpretations of omb enhancer trap patterns, the spatial distribution of Omb is not described by a step function but rather decays smoothly toward the periphery of the wing pouch. The graded Omb distribution precludes the occurrence of spatial Omb concentration discontinuities which cause abnormal cell shape and cell extrusion in the wing disc epithelium. Moreover, the Omb gradient appears to be required to specify a gradient of cell affinity along the A/P axis.
Omb is expressed in a gradient
In order to determine whether the graded distribution of Omb is required for the correct architecture of the wing disc epithelium, three types of mosaic clones were generated, all causing disruptions of the smooth Omb concentration profile. In the first, Omb was induced laterally to a level comparable to the endogenous peak concentration. In the second, Omb was reduced, and in the third, Omb was overexpressed beyond the maximum endogenous level. In mosaic discs, cytoskeleton and apico-basal organization were visualized by phalloidin staining (specific for filamentous actin), anti-α-tubulin staining (revealing the AMW), and anti-DE-cadherin immunofluorescence (revealing adherens junctions ).
Expression of constitutively active Tkv causes cell retraction in the lateral wing disc
The wing pouch AMW depends on Dpp signaling. AMW density is graded along the A/P axis with a broad maximum in the center of the pouch and attenuation laterally  (Additional File 4). In clones lacking Tkv activity, the AMW is strongly reduced [15, 20]. However, the AMW was also lost in retracted cells both inside and outside of UAS-tkv QD clones (Fig. 2D1' and 2E), suggesting that its presence depends on the spatial continuity of Dpp signaling. To test whether it is the up-regulated Omb level, elicited by increased Dpp signaling (Fig. 2B), which causes the retraction of cells in the lateral wing pouch, UAS-ombRNAi was co-expressed with UAS-tkv QD . OmbRNAi essentially eliminated Omb under these conditions. Furthermore, the retraction of lateral cells was prevented by this regime (Fig. 2F and Additional File 2B). Thus, the up-regulation of Omb is required to mediate the re-organization of the epithelial architecture in lateral act5C>tkv QD clones.
Lack or strong reduction of Omb causes retraction of cells in the wing pouch
JNK-dependent cell death has been observed previously on either side of the border of clones disrupting the normal Dpp signaling gradient . Cellular retraction in omb clones appeared independent of cell death. Although caspase-3 positive cells were occasionally observed at the border of clones (Additional File 5A, A' arrow), many retracting clones showed no evidence of cell death (Additional File 5A-C, arrowheads) and still proliferated (Additional File 5D and 5E, arrowheads). The relationship between cellular retraction and JNK-dependent cell death was analyzed in more detail for Omb-overexpressing clones (see below). In the adult wing, the majority of omb clones manifested as clustered microchaetae restricted to either the dorsal or ventral leaflet, similar to the predominant phenotype of UAS-tkv QD clones. Occasionally, omb clones survived to adulthood as cyst-like structures located between the dorsal and ventral wing surfaces indicating complete retraction of mutant cells during the pupal stage. In both cases, retracting cells survived up to the stage of cuticle deposition (Fig. 3H).
Overexpression of ombcauses cellular retraction in both autonomous and non-autonomous ways
Omb affects cell affinity in a concentration-dependent manner
In a reverse approach, we expressed Omb to different levels in the notum region of the wing disc which contains little endogenous Omb . Flip-out clones were generated in which omb was expressed under the control of tubα1>Gal4 or act5C>Gal4 (the relative strength of these Gal4 drivers as monitored by UAS-GFP expression was 1:1.8). While control clones had irregular outlines (Fig. 5C), clones expressing Omb were rounder, with a SF value that increased with Omb level (Fig. 5D, E, H).
Both experiments indicate that the level of Omb controls cell affinity and that Omb can exert this control both in its endogenous domain (pouch) as well as in an ectopic setting (notum).
Dpp gradient interpretation in the larval wing pouch
Thresholds, in a strict sense, should lead to a sharp transition in gene expression from one cell to the next . This is, in fact, not observed for any of the early wing pouch targets ([11, 41–45]; this report). Similarly, in the well studied Xenopus embryo model, activin forms a gradient that initially leads to a graded distribution of the target protein Xbra which is refined to form sharp boundaries under the involvement of secondary factors [46, 47]. Dpp, via Sal and Omb, specifies the highly stereotypic positions of wing veins L2 and L5 but also in this case additional genes are involved [48, 49]. Recent work on gradient interpretation supports the notion that smooth gradients of a single morphogen may not suffice to specify sharp transitions in nuclear or cell specification, e. g. [44, 50–53]. These findings suggest that the concept of positional information  may not be valid in its simplest form [13, 55].
Most studies on omb as a Dpp target gene in wing develoment were performed with omb-lacZ or omb-Gal4 enhancer trap lines [35, 56]. These lines quite faithfully render the overall omb expression pattern but differ from endogenous omb in detail (Additional File 1). Differences in the steepness of graded gene expression patterns between direct (RNA in situ hybridization or protein immunofluorescence) and indirect measurements (enhancer trap) have been noted before (e.g. Dad , brk ). A systematic deviation will occur when β-galactosidase is monitored by histochemical staining (e.g. with the common chromogen X-gal). β-galactosidase is a homotetrameric protein that is only active in its oligomeric form . This will cause a sigmoid dependence of activity on protein concentration. Similarly, when Gal4 expression is monitored by UAS-reporter activity, synergistic binding of dimeric Gal4 to the UAS pentamer of standard pUAST derivatives  can cause a non-linear response .
Presence of a gradient of cell affinity
Transplantation experiments in developing insect wings suggest that cells within a compartment differ in cell-cell affinity. Cells at the same proximo-distal position have a similar P/D affinity value and intermingle to form a wiggly interface. In contrast, groups of cells transplanted to different proximo-distal positions will rearrange contacts and form a roundish patch thereby minimizing contact with the surrounding tissue .
We tested whether a gradient of cell affinity is present along the A/P axis in the Drosophila wing disc by analyzing the shape of omb mutant clones (Fig. 5). We found that omb mutant clones close to the A/P boundary had smooth borders, indicating that omb mutant cells sort out from neighboring wild type cells. With increasing distance from the A/P boundary, clone shape became progressively irregular. This, in the framework of the cell affinity model, suggests the existence of a gradient of cell affinity which is disrupted by omb clones. The gradient of cell affinity correlates with the level of Omb expression, indicating that Omb, at least in part, shapes this gradient. This was confirmed by expressing Omb to different levels in a tissue with little endogenous Omb.
The importance of being graded
In addition to Dpp itself, several gene products directly or indirectly downstream of Dpp are expressed in a graded manner (e. g. Tkv, Brk, Dad, Sal, Omb, Capricious (Caps) and Tartan (Trn)). Some of these are part of the Dpp signaling cascade (Tkv, Brk, Dad), Sal and Omb are nuclear effectors, Caps and Trn cell surface proteins. Furthermore, the density of the AMW is graded along the A/P axis. In tkv clones, the AMW is lost [15, 20]. We show here, that in and around large UAS-tkv QD clones, in which the Dpp pathway is constitutively active, the AMW is also strongly reduced in retracting cells but not in central non-retracting cells (Fig 2D1'), indicating that the reduction in AMW density is elicited by the apposition of cells strongly differing in Dpp signaling acitvity. AMW reduction in tkv QD clones indicates that AMW density is not only controlled by the Dpp level but is also subject to control which is levied by Dpp signaling discontinuities.
Dpp is required for wing disc growth and proliferation [1, 16, 64]. The uniform proliferation across the wing disc has been difficult to reconcile with the exponential shape of the Dpp gradient and with the finding that ubiquitous expression of Dpp or of Dpp pathway components can promote overgrowth (reviewed in ). According to a model proposed by Rogulja and Irvine, two Dpp-dependent growth promoting systems coexist in the wing imaginal disc, only one of which is responsive to the gradient of Dpp signaling . More recently, Basler and colleagues argued, that a gradient of Dpp signaling is not required for wing growth . In the latter model, Dpp requirement differs qualitatively for growth and patterning.
Graded gene expression appears required also along the orthogonal dorso-ventral (D/V) axis. Vestigial is expressed in a symmetrical gradient that decays away from the D/V boundary  and is required for patterning and growth control along the D/V axis. Vg gain-of-function clones induce JNK at the clone border which is more remote from the D/V boundary, indicating that JNK is activated by spatial discrepancy in Vg levels . Such clones (and their wild type twin spots) become larger with increasing distance from the D/V boundary and retract from the apical epithelial surface . Apparently, the creation of local discontinuities in Vg level leads to increased proliferation on both sides of the clonal border. The importance of a graded Vg distribution is underlined by the reduced size of both vg mutant and Vg overexpressing wings . A similar requirement for graded gene expression to ensure normal wing disc proliferation was shown for dachsous and four-jointed [71, 72]. Like clones mutant for factors downstream of DPP, which disrupt A/P-boundary-centered gradients, vg mutant clones, in which JNK-mediated apoptosis is suppressed, are extruded from the wing disc . Retraction and extrusion, like morphogenetic apoptosis  may be universal mechanisms for correcting disturbances in the graded expression of factors required for patterning and growth of the wing disc epithelium. The gradient of cell affinity may serve to stabilize patterns of positional information against fluctuations of the respective morphogen activity gradients.
In the field of developmental biology, positional information and morphogens are important concepts to understand how cellular fields can be patterned. The Drosophila wing imaginal disc is a well studied system in which the diffusible protein Decapentaplegic, expressed in a stripe along the anterior-posterior compartment boundary, leads to the nested expression of target genes (spalt, omb, vestigial). The nested expression patterns are thought to arise from different thresholds of gene activation. We show by quantitative analysis that Omb expression is graded along the anterior-posterior axis. Manipulations that introduce spatial discontinuity in the Omb level cause disruptions of epithelial morphology, indicating that the normal graded distribution of Omb is important for proper wing development. We furthermore provide evidence that the Omb gradient instructs the formation of a gradient of cell affinity which may reduce cell mixing in the compartment.
Stocks are described at http://flybase.bio.indiana.edu unless indicated otherwise. l(1)omb D4 and l(1)omb 3198 were used as omb null alleles . Transgenes: UAS-CD8-GFP, tubα1>CD2>Gal4, act5c>CD2>Gal4, UAS-tkv QD , UAS-ombRNAi-C4 , UAS-omb 4-3 , UAS-bsk DN  and UAS-p35 . Enhancer trap lines: hh P30  and omb P1 . Larvae were reared at 25°C or at the indicated temperature.
tkv QD clones: y w hsp70-Flp; act5c>CD2>Gal4/UAS-tkv QD
CD8-GFP clones: y w hsp70-Flp; UAS-CD8-GFP; act5c>CD2>Gal4/UAS-CD8-GFP
tkv QD CD8-GFP clones: y w hsp70-Flp; UAS-CD8-GFP; act5c>CD2>Gal4/UAS-tkv QD
tkv QD ombRNAi clones: y w hsp70-Flp; UAS-ombRNAi; act5c>CD2>Gal4/UAS-tkv QD
omb clones: y w hsp-GFP hsp70-Flp FRT19/omb 3198 FRT1 and y w hsp-GFP hsp70-Flp FRT19/omb D4 FRT19
UAS-ombRNAi clones: y w hsp70-Flp; act5c>CD2>Gal4/UAS-ombRNAi and y w hsp70-Flp; UAS-ombRNAi; act5c>CD2>Gal4
UAS-omb clones: y w hsp70-Flp; act5c>CD2>Gal4/UAS-omb and y w hsp70-Flp; tub>CD2>Gal4; UAS-omb
UAS-omb UAS-p35 clones: y w hsp70-Flp; UAS-p35; act5c>CD2>Gal4/UAS-omb and y w hsp70-Flp/UAS-p35; act5c>CD2>Gal4/UAS-omb
UAS-omb UAS-bsk DN clones: y w hsp70-Flp/UAS- bsk DN ; act5c>CD2>Gal4/UAS-omb
wildtype control clones: y w hsp-GFP hsp70-Flp FRT19/FRT19 and y w hsp70-Flp; act5c>CD2>Gal4
Omb-antiserum, first mentioned in a footnote in , was raised against His-tagged full-length Omb protein expressed from the bacterial vector pET15b (Novagen, Darmstadt, Germany) and purified by Ni2+ chelate chromatography. Rabbits were immunized by sub-cutaneous, mice by intraperitoneal injection of the antigen along with antibody multiplier (Linaris, Wertheim, Germany).
Imaginal discs dissected from third instar larvae were fixed and stained with rhodamine phalloidin (Invitrogen, Carlsbad, CA, USA) and the appropriate primary antibodies: Rat anti-Ci 2A1, 1:4 (gift from R. Holmgren, Northwestern University, Evanston, IL, USA), mouse anti-CD2 (1:2000) (Serotec, Oxford, UK), rabbit anti-Omb (1:1000), rabbit anti-GFP (1:2000) (Clontech, Mountain View, CA, USA), rabbit anti-cleaved-Caspase-3 (1:200) (Cell Signaling, Danvers, MA, USA), rabbit anti-PH3 (1:200) (Upstate Biotechnology, Lake Placid, NY, USA), rabbit anti-β-galactosidase, 1:2000 (Cappel, Abnova, Heidelberg, Germany), goat anti-DE-cadherin (1:200) (Santa Cruz, Santa Cruz, CA, USA), and mouse anti-α-tubulin (1:1000) (Sigma, Munich, Germany). Secondary antibodies used were: Anti-mouse FITC, anti-mouse Cy5, anti-rabbit FITC, anti-rabbit Cy5, and anti-goat Cy3 (1:100, Jackson Immuno Research. West Grove, PA, USA). Images were recorded on a confocal microscope. The plot profile of anti-Omb staining was measured using the Image-J program (NIH, Bethesda, MD, USA).
Imaginal disc cryosections
After secondary antibody staining, discs were re-fixed for 30 min in 4% paraformaldehyde, washed, and stored in 30% sucrose at 4°C overnight. Discs were oriented in Tissue-Tek (Sakura Finetek, Torrance, CA, USA), frozen and cut into 25 μm sections on a cryostat (Cryo-Star HM 560, Microm).
Clonal shape measurement
For determination of position and shape factor of clones, the A/P boundary was determined by Ci or hh-lacZ staining, area (A) and perimeter (L) of clones were measured and the shape factor (4ΠA/L2) was calculated. The clonal position relative to the A/P boundary was determined by measuring the distance of the center of the clone to the A/P boundary divided by the distance from the edge of the wing imaginal disc to the A/P boundary
We thank K. Basler, A. Kopp, H. Sun, and the Bloomington Stock Center for fly stocks, R. Holmgren for antiserum, E. Jost for fly care, and Christoph Lichi Rickert for being helpful throughout.
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