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. Author manuscript; available in PMC: 2012 Aug 1.
Published in final edited form as: Mech Dev. 2012 May 14;129(5-8):147–161. doi: 10.1016/j.mod.2012.05.001

odd-skipped genes and lines organize the notum anterior-posterior axis using autonomous and non-autonomous mechanisms

Steven J Del Signore a, Teru Hayashi, Victor Hatini a,b,*
PMCID: PMC3409347  NIHMSID: NIHMS379708  PMID: 22613630

Abstract

The growth and patterning of Drosophila wing and notum primordia depend on their subdivision into progressively smaller domains by secreted signals that emanate from localized sources termed organizers. While the mechanisms that organize the wing primordium have been studied extensively, those that organize the notum are incompletely understood. The genes odd-skipped (odd), drumstick (drm), sob, and bowl comprise the odd-skipped family of C2H2 zinc finger genes, which has been implicated in notum growth and patterning. Here we show that drm, Bowl, and eyegone (eyg), a gene required for notum patterning, accumulate in nested domains in the anterior notum. Ectopic drm organized the nested expression of these anterior notum genes and downregulated the expression of posterior notum genes. The cell autonomous induction of Bowl and Eyg required bowl, while the non-autonomous effects were independent of bowl. The homeodomain protein Bar is expressed along the anterior border of the notum adjacent to cells expressing the Notch (N) ligand Delta (Dl). bowl was required to promote Bar and repress Dl expression to pattern the anterior notum in a cell-autonomous manner, while lines acted antagonistically to bowl posterior to the Bowl domain. Our data suggest that the odd-skipped genes act at the anterior notum border to organize the notum anterior-posterior (AP) axis using both autonomous and non-autonomous mechanisms.

1. Introduction

The generation of functional tissues and organs requires the precise specification of differentiated cell fates across a field of cells. Early in development, organizers are established within fields of cells to control the expression of transcription factors both autonomously and non-autonomously in patterns that ultimately prefigure the formation of adult structures (Lawrence and Struhl, 1996). The Drosophila wing imaginal disc gives rise to both the wing blade and the body wall (notum), and serves as an excellent model to study tissue patterning. Most work on organizer function in the wing disc has focused on the coordination of growth and patterning of the wing primordium along its dorsoventral (DV), anteroposterior (AP), and proximodistal (PD) axes. By contrast, the notum lacks an obvious PD axis, suggesting that the mechanisms that coordinate growth and patterning of this structure are distinct from those in the wing. However, such mechanisms have not been well studied.

A gene network specifies the notum territory of the wing disc and progressively subdivides it along the mediolateral and AP axes. The zinc finger protein Spalt major (Salm) is both necessary and sufficient for notum induction and has been proposed to act atop this hierarchy (Grieder et al., 2009). Subsequently, activation of the Drosophila epidermal growth factor receptor pathway (EGFR) in the presumptive notum represses Wg (Baonza et al., 2000) and induces expression of the Iro-C homeodomain proteins to specify notum identity (Zecca and Struhl, 2002; Wang et al., 2000; Simcox et al., 1996; Diez del Corral et al., 1999). In parallel, the bone morphogenetic protein (BMP)-like signal Decapentaplegic (Dpp) emanates from a narrow posterior domain to promote expression of the LIM homeodomain protein Tailup (Tup) and reinforce notum specification (Cavodeassi et al., 2002; de Navascués and Modolell, 2007; de Navascués and Modolell, 2010).

Interestingly, several notum specification genes play key roles in subdividing the mediolateral axis. Dpp promotes the expression of the GATA and FoG genes pannier (pnr) and u-shaped (ush) in the medial notum, (Fromental-Ramain et al., 2008; García-García et al., 1999; Letizia et al., 2007) where they coordinately promote proper bristle patterning (Cubadda et al., 1997, Sato and Saigo, 2000; Tomoyasu et al., 2000). Conversely, Dpp restricts expression of Iro-C genes to the lateral notum, where they specify the identity of this region. Dpp also acts in concert with the Pax-homeobox protein Eyegone (Eyg) to restrict Hth to the lateral notum to further elaborate the notum mediolateral axis (Aldaz et al., 2005).

In contrast to the patterning of the notum mediolateral axis, which is regulated positively by Vn and both positively and negatively by Dpp, the patterning systems that organize the notum AP axis are less well defined. The notum is subdivided morphologically into the anterior prescutum, the central scutum, and the posterior scutellum (see Fig. 1F). Each region bears a distinct pattern of sensory bristles comprised of repeated rows of many small bristles called microchaete, and the stereotyped placement of 22 larger macrochaete. Two gene families have been characterized to subdivide the notum AP axis. The homeodomain proteins Bar-h1 and Bar-h2 (hereafter referred to collectively as Bar) are expressed in the presumptive prescutum, where they are required to promote the correct pattern of sensory bristles. Bar mutant clones within the prescutum lack microchaete, and result in the elimination of the prescutal macrochaeta (Sato et al., 1999). The Pax-homeobox protein Eyg accumulates more broadly than Bar, both in the prescutum and in the scutum. Loss of eyg function leads to a severe reduction in size of the scutum, and a complete lack of both micro- and macrochaete. Conversely, overexpression of either eyg or its homolog twin of eyegone (toy) transforms the scutellum into scutum (Aldaz et al., 2003).

Figure 1. Nested expression of Bowl relative to drm and odd along the anterior border of the notum.

Figure 1

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(A–C) Pattern of Bowl accumulation during notum development. (A) Late second instar; Bowl accumulates in a broad anterior domain. (B) Third instar; the Bowl domain is limited to the anterior border of the notum. (C) 6hr APF; this pattern of Bowl accumulation persists into pupal stages. (D–E) Third instar; the Bowl domain (red) is slightly broader than the drm domain (green, D), and odd>RFP domain (green, E) Broader Bowl accumulation marked by arrowheads in insets. Note that the tissue curvature obscures much of the drm in situ signal, which accumulates apically (see Z sections inset; extent of drm signal marked by broad arrowheads). Note also that posterior expression of drm is in the overlying cuboidal epithelium, not the disc proper. (F) Schematics depicting the Bowl, drm, and odd domains in the notum at third instar (left) and pupal (right) stages. In this and all subsequent images, boxes indicate magnified regions shown in insets, arrows along the left side of an image indicate plane of Z section shown in corresponding insets. Scale bar indicates 20 μm in all panels.

Though Bar and Eyg provide mechanistic insight into the AP subdivision of the notum, the cues that elaborate their relative AP expression are not completely understood. It is clear that Bar expression in the lateral prescutum requires wg function, and that eyg expression requires both pnr and iro-C activity. However, these genes are expressed in mediolateral domains, and as such provide no AP patterning cues. Though Dpp secreted from the posterior notum restricts the expression of both Bar and eyg, the instructive cues that initiate the expression of Bar and eyg to promote anterior notum patterning remain unknown.

The C2H2 zinc finger odd-skipped (odd) family of genes is comprised of odd, bowl, sob, and drumstick (drm), and plays key roles in patterning both embryonic and larval tissues. Drm acts atop a relief-of-repression hierarchy in which Drm interacts with the protein Lines to prevent Bowl degradation (Green et al., 2002; Hatini et al., 2005). In the embryo, this pathway specifies alternative cell fates, depending on whether Bowl is active or repressed. In the hindgut, Lines and Drm/Bowl act in adjacent cell populations to specify small versus large intestine fates (Green et al., 2002; Iwaki et al., 2001; Johansen et al., 2003). Similarly, Lines and Drm/Bowl act antagonistically in the dorsal epidermis to specify 4° versus 1°–3° cell fates (Hatini et al., 2000; Hatini et al., 2005), and in the testis to specify somatic stem cell versus hub cell fates (DiNardo et al., 2011).

Though odd-skipped genes and lines have been studied in a variety of larval tissues, their developmental role appears more complicated than in the embryo. In imaginal discs, odd-skipped genes play both essential and redundant patterning roles, depending on context. In the margin of the eye disc, odd-skipped genes redundantly promote Bowl accumulation to promote hedgehog (hh) and trigger firing of the morphogenetic furrow (Bras-Pereira et al., 2006). In the leg, bowl is required to specify the distal tarsus (de Celis Ibeas and Bray, 2003), while the odd-skipped genes act redundantly to promote segmentation (Greenberg and Hatini, 2009; Hao et al., 2003). In the wing imaginal disc, lines represses bowl in most of the disc proper to allow normal wing development and PD patterning (Benitez et al., 2009). Conversely, the odd-skipped genes are expressed in and required for the specification of the squamous peripodial epithelium (PE) that overlies the disc proper (Nusinow et al., 2008). Interestingly, we found that in addition to the PE, odd-skipped genes were also expressed in the disc proper at the anterior border of the notum, in the presumptive prescutum. Broad expression of lines or bowlRNAi blocked the specification of the PE, the growth of the entire wing disc, and notably the formation of the notum (Nusinow et al., 2008). This phenotype, together with the expression of odd-skipped genes at the anterior border of the notum, suggested a role for these genes in patterning the notum AP axis

Here we investigated the role of the odd-skipped genes and lines in notum patterning. We find that drm, Bowl, and Eyg are expressed in nested domains along the anterior notum. drm was sufficient to promote accumulation of Bowl and Eyg both autonomously and non-autonomously. While the cell-autonomous induction of Eyg strictly required bowl function, drm was sufficient to induce Eyg non-autonomously independent of bowl. Subsequently, bowl was required to promote Bar and restrict Dl expression to pattern the anterior prescutum cell-autonomously. lines acted reciprocally to bowl to inhibit expression of anterior genes and promote expression of posterior genes. We propose that the odd-skipped genes establish an organizing center along the anterior border of the notum to promote expression of anterior notum genes and repress expression of posterior genes.

2. Results

2.1 odd-skipped family members are expressed in distinct patterns in the developing notum

To understand the roles of odd-skipped genes in notum development, we followed the pattern of Bowl accumulation throughout notum development and compared it to that of odd-skipped family members odd and drm (Fig. 1). By the late second instar Bowl accumulated in the squamous peripodial epithelium (PE), and in a broad anterior region of the notum (Fig. 1A). At the third instar, Bowl was limited to the anterior border of the notum in the presumptive prescutum (Fig. 1B), and this pattern persisted into pupal development (Fig. 1C). We compared the pattern of Bowl protein accumulation to drm mRNA expression at late third instar and found that Bowl accumulated more broadly than drm (Fig. 1D). The nested pattern of Bowl and drm expression was most pronounced in the lateral prescutum (Fig. 1D, arrowheads indicate the extent of the drm domain; arrows point to the limits of the broader Bowl domain). The broader accumulation of Bowl was surprising given that Drm has been considered to stabilize Bowl cell-autonomously by outcompeting the interaction between Lines and Bowl (Green et al., 2002; Hatini et al., 2005). Likewise, the Bowl domain was broader than the odd domain, marked by odd-GAL4 driving expression of a RFP reporter (Fig. 1E). The relative pattern of drm, odd, and Bowl expression at the late third instar and in the mature notum is summarized schematically in Figure 1F.

To better understand the dynamics of odd-skipped gene expression in the notum, we compared the accumulation of Bowl with both Eyg and mirror (mirr), both of which are required for notum specification and patterning (Fig. 2). mirr is required for notum specification and later patterning of the lateral notum (Cavodeassi et al., 2001), while eyg is required to specify the scutum (Aldaz et al., 2003). mirr is initially expressed broadly in the notum and becomes restricted to the lateral notum, while eyg is expressed broadly within the scutum. At the late first instar, Bowl and a mirr-lacZ reporter were expressed in largely complementary domains, with one to three rows of cells expressing both markers (Fig. 2A, arrowheads in insets point to overlap in expression). It is plausible that cells that co-express Bowl and mirr-lacZ at this stage are recruited to form the anterior border of the notum. Eyg was not expressed at this stage, suggesting that it acts downstream of bowl and/or mirr. At the late second instar, Bowl accumulated along the anterior border of the notum, while Eyg accumulated in a broader anterior domain. At this stage mirr-lacZ was repressed in the medial notum near the disc stalk and in the lateral prescutum (Fig. 2B). These relative patterns of gene expression were largely intact at the late third instar. Bowl, Eyg, and mirr-lacZ were co-expressed across several cell diameters along the anterior border of the notum, though mirr-lacZ was repressed entirely within the medial notum (Fig. 2C). The distinct expression patterns of Bowl and mirr-lacZ suggested that the two genes act in parallel pathways. In contrast, the broader nested accumulation of Eyg relative to Bowl suggested that the odd-skipped genes might promote notum expansion through both the autonomous and non-autonomous induction of Eyg.

Figure 2. Evolution of Bowl accumulation relative to mirr and Eyg.

Figure 2

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(A) 36–48 hr wing disc; Bowl (red) and mirr-lacZ (green) are detected in the notum prior to Eyg (blue/white). Bowl and mirr-lacZ expression overlap across 1–3 cell diameters (see arrowheads). (B) 72–84 hr notum; Bowl, mirr-lacZ, and Eyg are all expressed at this stage (arrowheads note nuclei expressing all markers in B and C). Bowl is largely restricted to the anterior margin, though it accumulates in a slightly broader domain across the medial notum (see inset). Eyg accumulates more broadly in the presumptive prescutum. mirr-lacZ is expressed broadly throughout the notum, except in the medial and antero-lateral regions. (C) These relationships are maintained at the third instar, though Bowl is more highly restricted to the prescutum, and mirr-lacZ is completely excluded from the medial notum (arrowhead marks medial boundary of expression). In this and all subsequent panels, all stains marked blue in merged images are shown as white in corresponding insets for clarity.

2.2 odd-skipped genes are required for notum formation at early first instar

We previously showed that broad expression of bowlRNAi or lines resulted in a severe reduction of wing growth and in a near complete loss of the notum. Interestingly, dpp expression was maintained in these discs suggesting that additional signals whose activities depend on odd-skipped genes’ function were required to promote notum growth and patterning (Nusinow et al., 2008). To better understand the role of lines and bowl at early stages of notum development, we expressed lines with the peripodial-specific driver Ubx-GAL4 (Fig. 3B, compare to wild type in 3A) (Nusinow et al., 2008). In addition, we generated large patches of lines-expressing clones at early larval stages (Fig. 3C). These manipulations resulted in adult flies lacking either one or both heminota (Fig. 3B,C; ΔT indicates missing thorax). This phenotype could arise from a loss of peripodial epithelium required for disc eversion at metamorphosis (Agnès et al., 1999), or from earlier defects in notum specification or growth. To distinguish between these possibilities, we examined the accumulation of Eyg in Ubx>bowlRNAi discs (Fig. 3E). In wild type discs, Eyg accumulates broadly in the anterior notum, and in restricted patches in the hinge and posterior wing pouch (Fig. 3D). Expression of bowlRNAi with Ubx-GAL4 severely downregulated Eyg accumulation in the notum (Fig. 3E), but did not affect accumulation in the pouch and hinge (Fig. 3E, asterisks). Broad overexpression of lines with C311-GAL4 eliminated expression of the homoedomain proteins C15 and Aristaless (Al), which are normally restricted to the posterior notum. Further, broad ectopic lines also eliminated the zinc finger protein Stripe (Sr), which is normally expressed in several anterior patches (Supp. Fig. 1). We further found that mirr-lacZ was still expressed in similar discs generated by induction of lines expressing clones at early first instar (Fig. 3F). This observation indicates that lines does not inhibit the specification of the notum but the elaboration of the notum AP axis. We note that large bowl, odd, or drm mutant cell clones generated in a Minute background failed to recapitulate this phenotype (Fig. 3G, Supp. Fig 2), suggesting that two or more odd-skipped genes act redundantly to broadly pattern the notum AP axis. However, the identical lines gain-of-function and bowlRNAi phenotypes supports the hypothesis that an antagonistic relationship exists between lines and the odd-skipped genes during the early stages of notum patterning, and that the odd-skipped genes are required for notum expansion.

Figure 3. Roles of lines and bowl in notum specification.

Figure 3

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(A–C) Dorsal views of adult head [H], thorax [T], and anterior abdomen [A]. (A) Wild type. (B–C) Expression of lines with Ubx-GAL4 (B) or in early first instar clones (C) disrupted the formation of the adult notum or heminotum, respectively. (D, E) Expression of bowlRNAi with Ubx-GAL4 resulted in a loss or severe elimination of AP notum gene expression. (D) Wild type; Eyg accumulates in a broad anterior domain in the wild type notum. (E) Ubx>bowlRNAi; Accumulation of Eyg in the notum was severely reduced. Only a small region expressed Eyg at low level near the disc stalk (compare bar to wild type). By contrast, Eyg accumulation in the pouch and hinge (marked with asterisks) was not affected. (F) In a disc bearing large patches of lines expressing FLP-out clones, mirr-lacZ expression was largely retained. (G) Early large Minute bowl clones did not adversely affect notum development, nor did large Minute odd or drm mutant clones (see Supp. Fig. 3).

2.3 odd-skipped genes and lines regulate Bowl accumulation in the developing notum

The bowlRNAi employed (Fig. 3E) is predicted to have off-target effects through partial complementarity with odd and sob transcripts. Thus, the bowlRNAi phenotype is likely to reflect the combined depletion of several odd-skipped genes. To test whether the odd-skipped genes can act redundantly, we examined the regulatory relationships between odd-skipped genes and lines in the notum and in vitro. As expected, ectopic drm expression in FLP-out clones resulted in a cell-autonomous stabilization of Bowl. Consistent with the endogenous pattern of drm and Bowl expression, Bowl was also stabilized non-autonomously surrounding the clones (Fig. 4A). By contrast, though sob and odd each promoted Bowl accumulation cell-autonomously, neither gene promoted non-autonomous accumulation of Bowl (Fig. 4B,C). Interestingly, we noted that Sob promoted Bowl accumulation to a much greater extent than did Odd. Together, these gain-and loss-of-function analyses suggest that each of the odd-skipped genes is sufficient to promote Bowl accumulation, though to varying degrees. Loss-of-function analysis revealed normal Bowl accumulation in drm and odd mutant clones (Fig. 4D,E), also supporting the hypothesis that two or more odd-skipped genes act redundantly to stabilize Bowl in the notum.

Figure 4. Regulation of Bowl accumulation by drm, odd, sob and lines in the notum and S2 cells.

Figure 4

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All clones are positively marked with GFP (green), except in G where clones are negatively marked by loss of GFP. (A) Ectopic drm resulted in both autonomous and periclonal (arrowheads) induction of Bowl (red in all panels in figure). (B–C) Ectopic sob (B) or odd (C) led to autonomous, but not periclonal, Bowl accumulation. (D–E) Neither drm (D) nor odd (E) mutant clones resulted in loss of Bowl accumulation. (F) Upper panel: Co-immunoprecipitation between FT-Bowl, HA-Drm, HA-Odd or HA-Sob and MT-Lines. Asterisks indicate mutant variants with a disrupted N-terminal zinc finger. MT-Lines formed a complex with each Odd family member containing a functional N-terminal zinc finger. Middle panel: Increasing levels of HA tagged Drm, Odd, or Sob outcompeted the interaction of MT-Lines with FT-Bowl. Lower panel: FT-Bowl accumulated at low levels in S2 cells (lane 1). Co-transfection with Myc-Lines further suppressed Bowl levels (lane 3), while co-transfection of Drm with Lines reversed this effect (lane 4). Co-transfection of Bowl with Drm (lane 2), Odd (lane 5) or Sob (lane 6) increased Bowl levels. (G) Bowl was lost along the presumptive prescutum in lines expressing clones, while it accumulated ectopically in lines mutant clones posterior the prescutum (H). (I) Schematic of functional interactions between Odd-skipped proteins and Lines. Thickness of arrows schematically suggests relative strength of interactions.

To explore the molecular mechanism by which the Odd-skipped proteins promote Bowl accumulation, we examined their interaction with Lines by co-immunoprecipitation assays in Schneider 2 (S2) cells. We found that Odd and Sob could each form a complex with Lines that required an intact N-terminal zinc finger domain (Fig. 4F, upper). Increased levels of Odd or Sob outcompeted the binding of Lines to Bowl (Fig. 4F, center), and enhanced Bowl accumulation (Fig. 4F, lower). Consistent with our overexpression studies, Odd showed a weaker capacity to bind to Lines and outcompete the binding of Lines to Bowl than did either Drm or Sob. In control experiments, we confirmed that Lines binds to Bowl, and that Drm outcompetes this interaction to stabilize Bowl as previously described (Fig. 4F) (Hatini et al., 2005; Green et al., 2002). These results suggest that Drm, Odd, and Sob could redundantly stabilize Bowl by inhibiting the interaction of Lines with Bowl. We then examined the regulatory relationship between Lines and Bowl in the notum. We found reduced Bowl accumulation in lines-expressing FLP-out clones within the Bowl domain (Fig. 4G). Reciprocally, we detected ectopic Bowl accumulation in lines FLP/FRT mutant clones outside the Bowl domain (Fig. 4H), but no accumulation surrounding the clones. Taken together these data confirm that Drm, Odd, and Sob can each bind and inhibit Lines to promote Bowl accumulation in vitro and in vivo. The regulatory interactions between Lines and the Odd-skipped proteins are summarized in Fig. 4I.

2.4 drm promotes Bowl and Eyg accumulation using cell-autonomous and non-autonomous mechanisms

Given the nested domains of expression of drm, Bowl, and Eyg, we tested by gain- and loss- of function analysis whether the odd-skipped genes are sufficient to mediate notum growth and patterning (Figs. 5 & 6). First, we asked whether ectopic expression of drm could promote anterior notum patterning. Indeed, we found that ectopic drm expression along the AP compartment boundary with dpp-GAL4 generated ectopic anterior notum structures, and led to a reduction of the posterior scutellum (Fig. 5B). In discs, this corresponded to a broad expansion of anterior fate marked by Eyg that coincided with the retraction of posterior fate, marked by Tup (Fig. 5D compare to wild type pattern in C). Additionally, loss of lines in mutant clones led to the loss of Tup autonomously in the posterior notum (Fig. 5E), further supporting the antagonistic relationship between these genes.

Figure 5. Ectopic posterior expression of drm induces ectopic anterior notum and inhibits scutellum formation.

Figure 5

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(A–B) Dorsal views of adult nota. Expression of drm with dpp-GAL4 resulted in the generation of ectopic anterior notum structures (red arrows) and a reduction of the posterior scutellum (red bracket, compare to wild type in A). (C) Third instar dpp>GFP notum; Eyg (green) and Tup (red) mark broad anterior and posterior domains, respectively, with a small overlap in central notum. (D) Third instar dpp>drm notum; Eyg was expanded ectopically into the posterior notum, while Tup expression was strongly diminished (expression remained in the overlying cuboidal epithelium, marked with an asterisk). (E–E′) Tup (red) was downregulated in lines mutant clones (negatively marked) within the Tup domain.

Figure 6. odd-skipped genes promote scutum expansion autonomously and non-autonomously.

Figure 6

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(A) drm overexpression in clones (green) posterior to the Eyg domain (blue/white) led to the autonomous and broad non-autonomous induction of Eyg (extent of non-autonomous induction outlined in white on main panel). (B) The autonomous, but not the non-autonomous, induction of Eyg was lost in drm expressing clones mutant for bowl. Apparent overlap in GFP and Eyg is due to projection (note lack of overlap in Z-sections). (C) In cases where clonal bowl-FLAG overexpression led to ectopic Bowl accumulation (anti-FLAG, red), it promoted autonomous expansion of Eyg accumulation. (D) Eyg (red) was induced autonomously in lines mutant clones posterior to the Eyg domain. (E–F) Eyg (red) was lost autonomously in early first instar bowl mutant clones (E) and in lines expressing clones (F, green). Arrowheads in E indicate Eyg positive nuclei surrounding the clone.

Clonal expression of drm promoted Eyg accumulation both within and broadly surrounding the clones, and Bowl accumulation both within and just adjacent to the clones (Fig. 6A). Thus, ectopic drm expression recapitulated the nested expression of these proteins in the notum. To determine whether bowl mediated the autonomous and non-autonomous activities of drm, we generated drm-expressing clones that were mutant for bowl, using the MARCM technique. We found that these clones failed to induce Eyg and Bowl accumulation cell-autonomously. However, these clones still induced accumulation of Eyg and Bowl non-autonomously (Fig. 6B). To test whether drm was sufficient to promote notum patterning outside the notum, we examined drm expressing clones in the pouch. As in the notum, drm expression induced Bowl both autonomously and non-autonomously (Supp. Fig 3A). Also similar to the notum, deletion of bowl in drm expressing clones abolished the autonomous, but not the periclonal accumulation of Bowl (Supp. Fig. 3B). However, in neither experiment did we detect autonomous or non-autonomous induction of Eyg, indicating that the outputs of drm activity depend on the tissue context. This observation is consistent with the finding that drm does not act to specify the notum, but rather to elaborate the AP pattern within the notum field (Fig. 3F).

To determine if bowl was sufficient to promote Eyg accumulation in the notum, we generated bowl overexpression clones using a strong UAS-bowl transgene. We found modest accumulation of Bowl and Eyg in a subset of clones, but none surrounding the clones (Fig. 6C). The weaker induction of Eyg in the Bowl expressing clones can be attributed to the lower level of Bowl that accumulated in these clones. Similar to bowl-expressing clones, lines mutant clones promoted accumulation of Bowl and Eyg only cell-autonomously (Figs. 4H and 6D, respectively).

To determine whether bowl was required to promote Eyg cell-autonomously, we examined Eyg accumulation in bowl mutant cell clones. We found that Eyg was downregulated in clones generated in the first larval instar (Fig. 6E). Eyg was also downregulated in lines expressing clones generated in the early first instar (Fig. 6F). By contrast, Eyg expression remained both in bowl mutant and in lines expressing clones induced after the first instar, suggesting that the maintenance of Eyg at the anterior border of the notum is independent of bowl (Supp. Fig. 4). Taken together, these results indicate that bowl is both necessary and sufficient to promote the cell-autonomous induction of Eyg, and that odd-skipped genes can organize notum growth and patterning using both autonomous and non-autonomous mechanisms.

2.5 bowl patterns the anterior prescutum cell-autonomously through the regulation of Bar and Dl expression

To further characterize the organization of the notum AP axis, we asked whether odd-skipped genes were required autonomously to specify the identity of the prescutum. We found that bowl mutant clones generated cuticle patterning defects within the bowl expressing prescutum (Fig. 7B, outlined in red). Expression of bowlRNAi or lines in the anterior notum with klumpfuss (klu)-GAL4 led to a significant reduction in the extent of the prescutum (Fig. 7C,D; Supp. Fig. 5C; p<.001). Additionally, microchaete were lost in the ventral prescutum (see arrows in Fig. 7C,D). This corresponded to a near total loss of Bowl accumulation in the prescutum (Supp. Fig. 5A–B). Since Bar genes (BarH1 and BarH2) are expressed in the presumptive prescutum and promote the patterning of this region (Sato et al., 1999), we asked whether the odd-skipped genes might promote Bar expression to specify the identity of this region. Consistent with the observed adult phenotypes, Bar-lacZ reporter expression was lost autonomously in bowl mutant clones (Fig. 7E), and similarly in clones of cells expressing either bowlRNAi or lines (Fig. 7F, G). Conversely, Bar accumulated ectopically in lines mutant clones (Fig. 7H). To further characterize the requirement of the odd-skipped genes in prescutum patterning, we examined the accumulation of Dl in the developing notum. Though N signaling has not been previously implicated in notum AP patterning, we noted that Dl accumulated adjacent to the Odd domain, with no accumulation in the presumptive prescutum (Fig. 7I). To further characterize whether loss of bowl altered the fate of the presumptive prescutum, we examined Dl expression in bowl mutant clones. We detected ectopic Dl in clones in the Bowl domain (Fig. 7J). Taken together, these results demonstrate that bowl is required autonomously to promote Bar expression and inhibit Dl accumulation to pattern the prescutum.

Figure 7. bowl specifies the prescutum.

Figure 7

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(A–D) Dorsal views of adult nota. (A) Wild type; red bracket demarcates prescutum. (B) Anomalies in cuticle differentiation (red outline) were observed in the prescutum of flies bearing bowl mutant clones. (C, D) Broad expression of bowlRNAi (C) or lines (D) with klu-GAL4 reduced the extent of the prescutum and eliminated lateral microchaete (red arrows). (E–G) Bar (shown by Bar-lacZ reporter) expression in the prescutum was lost in bowl mutant clones (positively marked, E), in bowlRNAi (F), and in lines (G) expressing clones. (H) Bar accumulated ectopically in lines mutant clones. (I) In control discs, Dl (red) accumulated adjacent to the odd domain (green, shown by odd>GFP). (J) Dl (blue/white) accumulated ectopically in the prescutum in bowl mutant clones (arrowheads delimit ectopic Dl accumulation).

During development, cells that adopt a particular cell fate minimize their interaction with cells of alternative fates. Cells that experimentally acquire a new fate frequently extrude from the epithelium or form compact structures to minimize contact with surrounding wild type cells (Shen and Dahmann, 2005; Villa-Cuesta et al., 2007). To determine whether the bowl mutant clones minimized contact with surrounding wild type cells, we analyzed their circularity, roundness, and solidity compared to wild type twin spots. We found that bowl mutant clones that were induced within the Bowl domain adopted a distinctly round and compact morphology in which actin accumulated apically (Fig. 8A). Mutant clones inside the Bowl domain were significantly different than clones outside the Bowl domain in all measures (p<.001, Fig. 8B). This phenotype suggests that the bowl mutant clones minimized contact with the surrounding Bowl expressing cells. Additionally, bowl mutant clones occasionally sorted from the Bowl domain and formed large composite clones with smooth borders and round morphology (Fig. 8C). The morphology and sorting behavior of the bowl mutant clones further support the hypothesis that bowl promotes cell fate within the prescutum.

Figure 8. bowl clones segregate from the Bowl domain.

Figure 8

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(A–B) bowl mutant clones induced at third instar in a wild type background adopted a rounder morphology and tended to segregate from the Bowl domain. (A) Boxed regions show clones inside (box 1) and outside (box 2) the Bowl domain (red). As quantified (B), bowl mutant clones within the Bowl domain were significantly rounder (p<.05), more circular (p<.001), and more compact (p<.01) than clones outside the Bowl domain (see section 4.4 for details). Asterisk marks twin spot clone within Bowl domain, which possesses normal morphology. (C) An example of a large composite clone at the edge of the Bowl domain.

3. Discussion

3.1 Epithelia are frequently patterned by signals from opposing field boundaries

In many developmental processes, signals that emanate from field borders play a crucial instructive role in patterning morphogenetic fields. The early Drosophila embryo is patterned by opposing gradients of Bicoid and Nanos that are generated from localized translation of corresponding mRNAs at the anterior and posterior poles of the embryo (Hoch and Jäckle, 1993). In the embryonic epidermis, the pattern of cell differentiation across each segment is regulated by the secreted Wg and Hh signals that emanate from localized sources at the anterior and posterior borders of each segment (Hatini and DiNardo, 2001). Similarly, the dorsoventral axis of the vertebrate spinal cord is organized by Shh ventrally, and BMP and Wnt signals that emanate from localized dorsal sources (Wilson and Maden, 2005; Gomez-Skarmeta et al., 2003). By contrast, current models of notum AP patterning focus mainly on the organizing influence of Dpp, which is secreted from the posterior border of the notum. We previously found that odd-skipped genes are expressed along the anterior border of the notum, and that broadly inhibiting their function in early wing discs caused a severe reduction or complete loss of the notum. As this reduction occurred despite the maintenance of dpp expression (Nusinow et al., 2008), we investigated whether the odd-skipped genes might define a second organizing center within the developing notum. Our current findings indeed suggest that signals that emanate from the anterior border of the notum act reciprocally to Dpp to promote expression of anterior notum genes and repress expression of posterior genes (Fig. 5–6). Through loss- and gain-of-function clonal analyses, we demonstrate that the odd-skipped genes pattern the notum AP axis both locally through regulation of Eyg, Bar, and Dl, and broadly through the regulation of Eyg and Tup. Finally, we show that lines acts antagonistically to bowl in this process (see model of the gene regulatory network in Fig. 9).

Figure 9. Model of the role odd-skipped genes in notum AP patterning.

Figure 9

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Schematic of notum AP gene expression patterns and regulatory relationships. Odd-skipped genes are abbreviated OSG. Note that Bar, OSG, and Eyg overlap in the prescutum, and that Dpp and Tup overlap in the posterior notum. Bowl accumulates in a slightly broader domain than drm and odd in the prescutum. There, Bowl is required autonomously for Eyg accumulation at the first instar, while drm acts redundantly with other odd-skipped family members to induce Eyg non-autonomously by unknown mechanisms. Further, odd-skipped genes autonomously stabilize Bowl in the prescutum to promote Bar and inhibit Dl expression to pattern this region. Dpp diffuses from the posterior to inhibit anterior genes Eyg and Bar (Aldaz et al., 2003; Sato et al., 1999).

3.2 odd-skipped genes pattern the notum AP axis using autonomous and non-autonomous mechanisms

We found that drm overexpression was sufficient to promote Eyg accumulation non-autonomously within the notum. This activity suggests that drm controls expression of an unidentified signal that spreads from the drm domain to induce Eyg accumulation non-autonomously. Alternatively, drm could initiate the propagation of a cascade of local inductive interactions to induce Eyg at a distance. Recent studies have shown that recruitment of cells to the wing field is accomplished by the propagation of a feed forward signal from the DV compartment boundary (Zecca and Struhl, 2007; Zecca and Struhl, 2010). In this process signaling at the border between Vestigial (Vg) and non-Vg expressing cells is used to recruit non-Vg expressing cells to the expanding wing field, a process dependent on signaling through the Fat-Dachsous pathway. Though we have yet to characterize a functional relationship between odd-skipped genes and Ft-Ds signaling, it is interesting to note that Ds accumulates in a complex graded AP pattern across the notum, consistent with such a role (SD & VH, unpublished observations).

In addition to the broad induction of Eyg accumulation, we were surprised to find that drm overexpression also induced Bowl in cells just adjacent to clones. Though the effect was subtle, we note that this pattern of activation recapitulated the endogenous nested pattern of drm and Bowl expression in the presumptive prescutum. It is unclear whether the nested expression of odd-skipped genes plays a functional role in notum AP patterning. Despite this, the concordance of endogenous and ectopic expression patterns supports the hypothesis that ectopic drm induces a physiologically relevant program of anterior gene expression in the notum. One possible clue as to the relevance of this nested pattern may come from the observation that only drm was able to promote Bowl non-autonomously. In contrast, lines−/−, odd+, and sob+ clones each induced only cell-autonomous accumulation of Bowl. Notably, these clones rounded up and segregated from the epithelium, while drm expressing clones remained integrated with the surrounding epithelium. One interpretation of these data is that abrupt discontinuities in the level of Odd-skipped proteins may alter epithelial morphology, as previously reported (Hao et al., 2003). This interpretation is further supported by the observation that bowl mutant clones within the Bowl domain adopt a compact, round morphology relative to clones outside the Bowl domain (Fig. 8). We hypothesize that drm promotes lower levels of Bowl in nearby cells to dampen otherwise sharp discontinuities in Bowl activity to regulate local buckling of the epithelium.

Alternatively, differences in the total levels or ratios of Odd family proteins along the anterior border of the notum could elicit different transcriptional outcomes. Since Odd and Bowl have been shown to interact with the transcriptional co-repressor Groucho, variation in the levels of the Odd-skipped proteins could titrate Groucho and affect Groucho-dependent transcriptional outputs (Benítez et al., 2009; Goldstein et al., 2005). Alternatively, given their distinct structure outside the zinc finger domain, the Odd-skipped proteins could interact with distinct sets of target genes to pattern the anterior border of the notum. Though additional experiments will be required to ascertain whether such mechanisms are active in the prescutum, we provide evidence that bowl is strictly required for the early autonomous induction of Eyg, the later expression of Bar genes, and the repression of Dl. These results provide evidence that odd-skipped genes act both independently and redundantly to organize the notum AP axis.

3.3 Redundant versus unique functions of odd-skipped genes in notum development

We show that bowl is essential for patterning the prescutum, but not for broadly patterning the notum AP axis. Previous studies have revealed a variety of essential and redundant functions for odd-skipped family genes in patterning embryonic and larval tissues. In the embryo, drm and bowl antagonize lines function to pattern the dorsal embryonic epidermis, foregut, and hindgut (Iwaki et al., 2001; Johansen et al., 2003; Green et al., 2002; Wang and Coulter, 1996; Lengyel and Iwaki, 2002), while odd functions as a pair rule gene to promote embryonic segmentation (Coulter et al., 1990; Coulter and Wieschaus, 1988). In the leg imaginal disc, bowl is essential for patterning the tarsal proximodistal axis at early stages, but acts redundantly with other odd-skipped genes to control leg segmentation later in development (de Celis Ibeas and Bray, 2003; Hao et al., 2003; Greenberg and Hatini, 2009). In the eye, bowl is essential for the initiation of retinogenesis from the eye margin (Bras-Pereira et al., 2006), while odd and drm have been proposed to activate Bowl redundantly.

Our loss-of-function analysis revealed that neither drm nor odd is necessary to stabilize Bowl. At present we cannot exclude the possibility that sob is necessary to promote Bowl accumulation because a null sob mutant is not yet available. Our biochemical and genetic analysis demonstrates that not only Drm, but also Odd and Sob can each outcompete the interaction of Lines with Bowl and stabilize the Bowl proteins in S2 cells and in vivo. These results suggest that different combinations of Odd-skipped proteins could be used to activate bowl depending on context.

Previous work suggested reciprocal roles for lines and odd-skipped genes in subdividing the early wing disc into disc proper and peripodial epithelium. The loss-of-function analysis described in this study suggests that the odd-skipped genes act redundantly to control the early specification of the PE and the subsequent expansion of the notum, while revealing an essential role for bowl in specification of the anterior prescutum. Redundancy can increase the robustness of essential developmental processes and provide a buffer against fluctuations in activity of single genes. The redundant role of the odd-skipped genes in PE specification and notum expansion could therefore serve to ensure the optimal growth of the wing disc at early stages and that of the notum at later stages and protect these critical processes from perturbations.

3.4 Conclusion

The growth and patterning of the wing field are coordinated with the elaboration of the wing PD axis. The developing notum lacks an obvious PD axis, and instead is subdivided into a series of AP and mediolateral domains (Calleja et al., 2002). The establishment of organizers that act antagonistically from opposing field borders is a robust strategy to subdivide the notum AP axis. Our work demonstrates that the odd-skipped genes act autonomously at the anterior border of the notum to specify the prescutum, and non-autonomously at short and long range to control the expression of transcription factors that prefigure the differentiation of the notum AP axis. Though further experiments will be required to characterize the mechanism by which this putative organizer acts, our studies provide evidence that the anterior border of the notum exhibits the functional attributes of an organizer.

4. Materials and Methods

4.1 Fly strains and clonal analysis

FRT42D linesG2, bowl1 FRT40A, drm3 FRT40A, and oddrk111-lacZ FRT40A were used to generate mitotic mutant cell clones using the FLP/FRT (Golic, 1991; Xu and Rubin, 1993) and the MARCM techniques (Lee and Luo, 2001) at 48–72, 72–96 and 96–120 hours AEL, which correspond to second, early third and mid third instar. Flies of the genotype y w hs-FLP; FRT42D Ubi-GFP (B. Edgar) were used to generated FLP/FRT clones and y w hs-FLP Tub-GAL4 UAS-GFP-6Xmyc-NLS; FRT42D Tub-Gal80 hs-CD2, y+ (gift of G. Struhl) to generate MARCM clones. Ubi-GFP M(2)24F[1] FRT40A was used to induce mutant clones in a Minute background (gift of E. Moreno). wg-lacZ (Kassis et al., 1992), oddrk111-lacZ, bar-lacZ (gift of T. Kojima), mirrDE-lacZ, and dpp-lacZ were used to map domains of gene expression. UAS-Lines9.2 (strong insertion), UAS-Myc-Lines8 (weak insertion), UAS-Flag-bowl #21 (strong insertion), UAS-GFP (B. Edgar), UAS-bowlRNAi #3774 (VDRC) were expressed in clones using y w hs-FLP; act5C>y+>GAL4 UAS-GFP (Pignoni et al., 1997) or specifically in the anterior notum using klu-GAL4, and along the AP compartment border using ptc-GAL4 and dpp-GAL4. Note that odd and sob are off targets of the BowlRNAi due to partial complementarity.

4.2 Immunofluorescence and microscopy

Staining protocols have been described elsewhere (Hatini et al., 2005). Primary antibodies used were: mouse anti-Wg (4D4, DSHB) (Brook and Cohen, 1996), rabbit anti-Bowl (generated in this study after (de Celis Ibeas and Bray, 2003), rat anti-Al and rat anti-C15 (gifts of G. Campbell), guinea pig anti-Eyg (gift of N. Azpiazu) (Aldaz et al., 2003), mouse anti-FLAG M2 (Sigma), mouse anti-β-galactosidase (DSHB), guinea pig anti-Senseless (GP55, gift of H. Bellen) and guinea pig anti-Stripe (gift of T. Volk). Confocal images were acquired using a Zeiss LSM510 in multi-tracking mode. Immuno-in situ protocol was based on work described elsewhere (Rhiner et al., 2010). Briefly, Digoxigenin (DIG)-labeled probes were hybridized overnight at 55°C, detected with Peroxidase (POD)-anti-Dig followed by direct Tyramide signal amplification (TSA) Cy3 (Perkin Elmer) to generate a fluorescent signal. Bowl was then detected by indirect immunofluorescence using a standard protocol.

4.3 Interaction of Lines with Odd-skipped proteins in S2 cells

HA-Drm and Flag-Bowl constructs were previously described (Hatini et al., 2005; Green et al., 2002). Bowl constructs were generated from cDNA clone RE32660, Odd from clone RE57157, and Sob from clone RE2226. Drm (C28L), Odd (C222L), Sob (C397L) mutant variants were generated by substituting the first cysteine of the first conserved C2H2 zinc finger motif with a Leucine. Bowl (R258C) was generated by substituting an arginine in loop 3 of the first zinc finger with a cysteine. Variants of each of the odd-skipped family genes were generated by PCR amplification and fused in frame with corresponding tags in pCS2 2X-Flag, pCS2 6X-Myc, or pCS2-2X-HA to generate N-terminally tagged proteins. Primer sequences are available upon request. Three glycine residues separated the tags from the coding region. Following sequencing, tagged cDNA were cloned into either pUAST or pUASp. For analysis of the interaction of Lines with each of the Odd-skipped proteins and their mutant variants, S2 cells were transfected using calcium phosphate in a 6-cm dish with 3 μg of Ubiquitin-GAL4, 2.5 μg MT-Lines and 2.5 μg of wild type and corresponding mutant variant of each Odd-skipped protein. Immunoprecipitation assays were performed as previously described (Hatini et al., 2005; Green et al., 2002) using anti-Flag antibodies for Bowl (M2; Sigma) and anti-HA antibodies for Drm, Odd, and Sob (HA.11, Babco) at 1:40 dilution, followed by immunoblotting with rabbit anti-Myc (A-14; Santa Cruz) at 1:1000 dilution. The amounts of Myc-Lines in unprocessed lysates were used to normalize for variations in transfection efficiency. For competition assays, cells were transfected with 2.5 μg of Ubiquitin-Gal4 and 2 μg of UAS-Myc-Lines, in the presence of increasing amounts of wild type UAS-HA-tagged protein (0, 2 and 8 μg). Immunoprecipitation assays were performed using anti-Flag antibodies followed by immunoblotting with anti-Myc antibodies. For Bowl stabilization assays, cells were transfected with 3 μg of Ubiquitin-GAL4, 1 μg of MT-GFP, 2.5 μg MT-Bowl and 2.5 μg of MT-Lines and/or an HA-tagged Odd-skipped protein as indicated. MT-Bowl levels were detected in the lysates and normalized to MT-GFP levels.

4.4 Analysis of clone shape

bowl mutant clones were induced from 72–96 hrs AEL and dissected at late third instar. Discs were stained for Bowl and clones analyzed both inside and outside the Bowl domain (n=12 and 21, respectively). Clones were traced manually with ImageJ and analyzed using Shape Descriptors to measure the roundness, circularity and solidity of the clones. Microsoft Excel was used to perform t-tests with a Bonferroni correction for multiple tests to determine differences between groups. Circularity = 4 π *(area/perimeter2). A value of 1.0 indicates a perfect circle; Roundness = 4*area/(π *major axis2); Solidity = area/convex area.

4.5 Thorax measurements

Adult flies were photographed using a Fuji FinePix digital camera mounted on a Zeiss Stemi SV11 stereomicroscope and stacks were processed using CombineZP to generate in-focus composite images (A. Hadley, available: http://www.hadleyweb.pwp.blueyonder.co.uk/). The lengths of thorax subdomains were measured along the dorsal midline from the anterior-most bristle to the anterior limits of the prescutal suture, the scutellar suture, and the posterior limit of the scutellum using ImageJ (NIH, http://rsbweb.nih.gov/ij/). Microsoft Excel was used to perform t-tests with a Bonferroni correction for multiple tests to determine differences between groups.

Supplementary Material

Acknowledgments

We thank S. Bray, G. Campbell, K. Irvine, T. Kojima, R. Mann, E. Moreno, P. Mitchell, C. Rauskolb, G. Struhl, B. Edgar, T. Volk, N. Azpiazu, U. Walldorf, H. Bellen, the Bloomington Stock Center, the Vienna Drosophila Research Center (VDRC), and the Developmental Studies Hybridoma Bank for generous gifts of fly stocks and antibodies. We also thank D. Nusinow, K. Wharton, and members of the lab for comments on the manuscript. This work was supported by a grant from the NIH to V.H. (R01GM06806).

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NIHMS379708-supplement-supplement_1.pdf (1.3MB, pdf)

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