Clonal analyses

Clones were induced by hs-flp using the FLP/FRT technique (Xu and Rubin, 1993). To provide mutant cells with a growth advantage, we employed the Minute technique, which allows mutant cells to grow faster than surrounding heterozygous cells (Morata and Ripoll, 1975). For clonal analyses, we used both Stat92E^85C9 and Stat92E³⁹⁷, which are strong loss-of-function mutations (Ekas et al., 2010), and obtained similar results. Larvae were heat shocked for 2 hours at 39°C during first or second instar, unless otherwise specified. Since the tsh gene lies in close proximity to the FRT⁸⁰ site, we generated tsh loss-of-function flip-out clones using a hairpin construct (UAS-tshi) and UAS-lacZ to mark them. We induced AyGal4 flip-out clones (Ito et al., 1997) by heat shocking for 2 hours at 39°C. Ectopic expression was also achieved with the Gal4/UAS technique (Brand and Perrimon, 1993). Adult wings were dissected and mounted in a 2:l mixture of Canada balsam and methyl salicylate.

Stat92E functions in parallel to Hth in regulation of the hinge

To determine the fate of cells with activated Stat92E in the adult wing, we used the 3XGrh/4XdSTAT-LacZ reporter, which contains four multimerized Stat92E binding sites regulating expression of the lacZ gene (Tsai et al., 2007). Indeed, JAK/STAT signaling is localized to the adult hinge (Fig. 2A). Furthermore, wings with Stat92E clones display a near complete lack of hinge structures and are directly attached to the thorax and locked in perpendicular alignment to the body wall hindering their ability to flutter (Fig. 2B and not shown).

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Figure 2

Stat92E is critical for hinge development

(A) Expression of the 4xdSTAT-lacZ reporter in the adult wing hinge (blue) shows that JAK/STAT pathway activity remains active in the hinge until adulthood.

(B) Stat92E clones lead to loss of hinge structures (arrow) resulting in a wing blade that connects directly to the thorax. Clones are unmarked in the adult.

(C, D) Stat92E activity (10xSTAT-GFP, green) is largely overlapping with Hth (blue) in an early (C) and middle third instar wing disc (D) in the hinge. However, Hth is expressed in the notum (C′, D′, arrows) whereas 10xSTAT-GFP is not (C″, D″, arrows). Wg is red is C and D. C′, D′ are single channels for Hth; C″, D″ for 10xSTAT-GFP.

(E, F) Hth protein (blue in E and magenta in F) is slightly increased in Stat92E that lack GFP in early (E) and late third instar (F) discs. Vg is red is E. Arrows in E′ and F′ indicate regions of elevated Hth protein. E′, F′ are single channels for Hth.

(G) Hth (red, arrow in G′) is not altered by ectopic activation of the JAK/STAT pathway in Hop flip-out clones residing in the notum (labeled actinhop, green). G′ is single channel for Hth.

(H, I) Hth (blue) is repressed in Hop flip-out clones (green) residing in the notum (H′, I′, red arrows). Hth can be induced in an ectopic growth domain induced by a Hop flip-out clone residing next to the wg-lacZ (red) domain in the notum (I′, green arrow). H′, I′ are single channels for Hth.

(J) Stat92E activation (green, arrow in I′) is lost in a hth clone (indicated by the loss of LacZ (red)). J′ is single channel for 10xSTAT-GFP.

(K, L) Stat92E activity (green) can be upregulated by Hth flip-out clones (red, labeled actinhth) that reside in within the pouch (K′, arrows) and the notum (L′, arrow). K′, J′ are single channels for 10xSTAT-GFP.

Hth is a key regulator of hinge fate (Azpiazu and Morata, 2000; Casares and Mann, 2000). Stat92E activity shares considerable overlap with Hth expression during third instar in the presumptive hinge (Fig. 2C–D″). However, Hth is expressed in the medial notum in third instar (Fig. 2C′, D′, arrows and (Azpiazu and Morata, 2000; Casares and Mann, 2000)), whereas 10xSTAT-GFP is not (Fig. 2C″, D, arrows). To determine if Stat92E impacts Hth expression, we examined Hth protein in Stat92E clones. Hth is slightly but consistently elevated in Stat92E clones at early and late stages of wing development (Fig. 2E′, F′, arrows), suggesting that Stat92E may negatively regulate Hth. We addressed whether ectopic activation of JAK/STAT signaling could alter Hth expression. We generated Hop-expressing clones, which activate Stat92E in a ligand-independent and cell-autonomous manner (Rodrigues et al., 2012). When Hop-expressing clones reside within the hinge, Hth expression is not changed (Fig. 2G′, arrow). By contrast, cell-autonomous reductions in Hth levels were observed in Hop expressing clones residing within the notum (Fig. 2H′, I′, red arrows). Finally, ectopic Hth is observed in Hop flip-out clones residing in the dorsal notum next to the wg stripe that induce re-patterning and an ectopic outgrowth (Fig. 2I′, green arrow). We next investigated how loss- of gain-of-function mutations in Hth affected JAK/STAT signaling. 10xSTAT-GFP and epithelial folds corresponding to hinge fate are lost in hth clones (Fig. 2J′, arrow), indicating that Stat92E is downstream of Hth in the hinge. Consistent with this observation, Stat92E activity is frequently increased in a cell-autonomous manner in Hth-expressing clones residing at the proximal edge of the pouch (Fig. 2K′, arrows). The upregulation of Stat92E activity in these clones never extends to the distal part of the clone, presumably because pouch factors like Nub and Rn (see below) repress Stat92E activity there. Lastly, ectopic 10xSTAT-GFP expression is observed within Hth-expressing clones encompassing the dorsal notum (Fig. 2L′, arrow). Similar to the pouch examples, the ectopic Stat92E activity in Hth-expressing clones within the notum is cell-autonomous but does not fill the clone boundary, presumably because notum factors repress it (see below). Taken together, we conclude that JAK/STAT signaling may exert a negative effect on Hth protein levels and that Hth - with its ability to induce Stat92E activity - resides at the top of the hinge regulatory network.

Stat92E induces hinge-specific genes and represses notum-specific genes

To determine the effect of Stat92E activity on hinge-specific genes, we monitored expression of Msh, ds and Zfh2 in clones with increased or decreased JAK/STAT signaling. In wild-type third instar discs, Msh exhibits considerable overlap with 10xSTAT-GFP in the hinge (Fig. 3A–A″). Zfh2 and ds have been previously reported to be localized to the hinge and Zfh2 is required for its development (Terriente et al., 2008; Whitworth and Russell, 2003; Zecca and Struhl, 2010). Consistent with Stat92E residing near the top of the hierarchy of genes controlling hinge development, Msh, ds and Zfh2 are reduced in a cell-autonomous manner in Stat92E clones residing in the presumptive dorsal or ventral hinge or pleura (Fig. 3B′, C′, E′, arrows). In addition, Msh is autonomously induced in Hop-expressing clones residing in the notum clearly away from the Msh endogenous expression domain (Fig. 3F–F″). Zfh2 seems to be induced by ectopic Stat92E activation in a more restricted manner, weakly and only in a limited part of Hop-expressing clones residing outside of the hinge (Fig. 3D′, arrows). One important function of Msh is to counteract the Iro-C protein Ara and they are expressed in complementary patterns (Fig. 3G and (Villa-Cuesta and Modolell, 2005)). Ara is ectopically expressed in the hinge region in Stat92E clones (Fig. 3H′, red arrow). This appears to be cell-autonomous because Ara is not ectopically expressed in neighboring heterozygous tissue that contains one functional Stat92E allele (Fig. 3H′, green arrow). We tested whether ectopic activation of JAK/STAT signaling could cell-autonomously repress Ara within its endogenous domain. Ara is autonomously repressed in Hop-expressing clones that activate JAK/STAT signaling (Fig. 3I′, arrows). In most instances, the loss of Ara within the clone is associated with upregulation of Msh (Fig. 3I′, I″, green arrow). However, occasionally we observed repression of Ara in Hop-expressing clones that do not have ectopic Msh (Fig. 3I′, I″, red arrow). We conclude that JAK/STAT signaling can repress Ara expression through Msh-dependent and Msh-independent means.

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Figure 3

Activated Stat92E induces hinge-specific genes and represses notum genes

(A) In a wild type mid- to late third instar disc, Stat92E activity (green) largely overlaps with that of Msh (red) in the proximal hinge. Wg is blue. A′ is single channel for Msh; A″ for 10xSTAT-GFP.

(B) Msh (red) is lost in a cell-autonomous manner in Stat92E clones (B′, arrow), which lacks GFP. Clone boundaries are outlined by dashed yellow lines. B′ is single channel for Msh; B″ for GFP.

(C) Zfh2 (magenta) is reduced in a cell-autonomous manner (C′, arrows) in Stat92E clones, which lack GFP. C′ is single channel for Zfh2.

(D) Zfh2 (magenta) is ectopically induced in a cell-autonomous manner within some regions of Hop-expressing clones, for example at the edges of clones residing in the notum (D′, arrows). D′ is single channel for Zfh2.

(E) ds-lacZ (magenta) is reduced in a cell-autonomous manner (E′, arrows) in Stat92E clones, which lack GFP. E′ is single channel for ds-lacZ; E″ for GFP.

(F) Msh (red) can be induced in a cell autonomous manner (F′, arrow) in Hop-expressing clones residing in the notum. Dlg is blue. F′ is a close-up of the Hop flip-out clone. F″ is the single for Msh.

(G) Msh (green) and Ara (red) have mutually exclusive expression patterns. Dlg is blue.

(H) In a Stat92E clone, Ara (red) expression is expanded into the into the lateral hinge (H′, red arrow). By contrast, Ara is not increased in neighboring heterozygous cells that contain one functional copy of Stat92E (H′, green arrow). H′ is single channel for Ara.

(I) In Hop-expressing clones (green), Ara (red) is repressed in a cell-autonomous manner (arrows in I′). Msh is blue. Most often Ara repression is associated with upregulation of Msh within the Hop flip-out clone (I″, I″, green arrows). However, less frequently, we observe repression of Ara in the Hop flip-out clone without increases in Msh (I′, I″, red arrows). I′ is single channel for Ara; I″ for Msh.

JAK/STAT pathway activity is required for growth of the gap domain

The gap domain is an independent growth domain formed in late second instar after Wg represses Tsh from the pouch (Zirin and Mann, 2007). Cells in the gap domain lack Nub and Tsh, and this domain expands from late second instar until the end of larval development. Stat92E activity is detected at high levels in cells within the gap domain (Fig. 1M′), raising the possibility that JAK/STAT signaling is required for its formation and/or maintenance. Consistent with the loss of the hinge domain in adults with Stat92E clones, we observed a cell-autonomous loss of the gap domain in Stat92E clones induced 48 hours AED (compare yellow line to white line in Fig. 4A–A ″). This domain is also reduced in Stat92E clones induced later in development at 96 hours AED (compare yellow line to white line in Fig. 4B–B). These data suggest a continuous requirement for JAK/STAT activity in growth of the gap domain. We addressed whether factors expressed in the hinge and at least partly regulated by JAK/STAT signaling are also required for formation or maintenance of the gap domain. We found that the gap domain in msh mutant clones is comparable to control tissue from the same disc (Fig. S3A–C). In addition, chinmo, which is regulated by JAK/STAT signaling in the eye disc (Flaherty et al., 2010), is not also required for maintenance of the gap domain, despite being expressed in the hinge (Fig. S3D–G). Thus, two factors that are expressed in the hinge and can be regulated by JAK/STAT signaling are not required for formation or maintenance of the gap domain.

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Figure 4

JAK/STAT signaling is required for growth of the gap domain

(A, B) Stat92E clones induced at 48 (A) or 96 (B) hours AED, marked by the absence of GFP, result in loss (A) or reduction (B) of the gap domain and juxtaposition of the Tsh (blue) and Nub (red) domains. xz sections are marked in (A, B) by white and yellow lines crossing heterozygous (A″, B″) and mutant (A, B) tissue, respectively. Nub and Tsh expression are shown together in A′ and B′.

(C) In wild type, the Wg OR (red) lies within the proximal region in cells that express high levels of JAK/STAT signaling (green). By contrast the Wg IR (red) abuts the distal end of JAK/STAT activity in the gap domain. C′ shows a close up of the area outlined by the yellow box in C. The region that was scanned as an xz section is marked by a white line in C′. C″ shows 10xSTAT-GFP, wg and Tsh (blue). C shows 10xSTAT-GFP and wg. Note the overlap of 10xSTAT-GFP with the Wg OR and mutual exclusion of 10xSTAT-GFP with the Wg IR.

(D, E) Stat92E clones in the hinge marked by the absence of GFP result in a proximal shift of the Wg IR (red) moving it closer to the Wg OR (also red) (D′, E′, arrow) compared to surrounding heterozygous tissue. xz sections of Wg (white) through heterozygous Stat92E tissue (white line) is shown in D″ and through Stat92E homozygous mutant tissue (yellow line) is shown in D. Note the collapse of the Wg IR onto the Wg OR in D. D′, E′ are single channels for Wg.

(F) The region between the Wg IR and OR (marked by wg-lacZ, red) is not increased in Hop flip-out clones (green) (F′, arrows). By contrast, wg in the notum is repressed by ectopic activation of the JAK/STAT pathway (F, green arrow). F′ is single channel for wg-lacZ.

(G, H) Within the gap domain, Stat92E mutant tissue, marked by the absence of GFP, contains fewer EdU-positive (blue) cells than heterozygous tissue, marked by the presence of GFP. wg-lacZ (red) marks the Wg IR and OR. A magenta line marks the Stat92E mutant tissue and a green line marks the heterozygous tissue located between the Wg IR and Wg OR (G′-G). G′ is single channel for GFP; G″ for wg-lacZ; G for EdU. Graphical representation of EdU-positive cells in Stat92E mutant (magenta bar) or heterozygous (labeled Min/+) (green bar) tissue in 10 wing discs. The mean of the number of EdU positive cells/pixels sq +/− the standard deviation of the mean is 0.0090 +/ 0.0006 in Min/+ tissue and 0.0061 +/− 0.00051 in Stat92E mutant tissue. Asterisks indicate a statistically significant difference between these genotypes (P<0.002).

The gap domain resides largely between the Wg IR and OR (Fig. 4C–C and (Zirin and Mann, 2007)). In wild type, Stat92E activity abuts the Wg IR and encompasses the OR (Fig. 4C). In Stat92E clones, the Wg IR shifts proximally and collapses into a single ring with the Wg OR, while the position of the OR is unaffected (Fig. 4D, arrows). By contrast, the distance between Wg IR and OR is unaffected in adjacent Minute/+ tissue (Fig. 4D″, arrows). The reduction in the distance between in the Wg IR and OR is not limited to the dorsal hinge but rather can be observed throughout the hinge (Fig. 4E′, green arrows). We next addressed if we could expand the gap domain or affect the Wg rings by ectopic activation of the JAK/STAT signaling pathway. We and others previously showed that ectopic Stat92E can repress wg in the eye, antennal and leg discs and in the stripe of wg in the notum of the wing disc (Ayala-Camargo et al., 2007; Ekas et al., 2006; Tsai et al., 2007). We find that ectopic JAK/STAT signaling in clones does not alter the Wg IR or OR and does not increase the size of the gap domain (Fig. 4F′, red arrows), consistent with our prior report (Rodrigues et al., 2012). We verified that our Hop flip-out clones had ectopic Stat92E activity because a Hop-expressing clone in the notum autonomously represses wg expression there (Fig. 4F′, green arrow). Therefore, we conclude that JAK/STAT signaling is necessary but not sufficient for the growth of the domain between the Wg IR and OR.

To assay directly if JAK/STAT signaling could regulate growth of the gap domain, we generated Stat92E clones after 96 hours of development in order to reduce (but not eliminate) the gap domain. We labeled the discs with EdU to mark cells that were replicating their DNA. We restricted our analysis to tissue located between Wg IR and OR in the dorsal hinge. We counted the number of EdU-positive cells between the Wg IR and OR in Stat92E tissue as compared to EdU-positive cells in adjacent Minute/+ control tissue. Fig. 4G is a representative example of the 10 discs we analyzed that had Stat92E mutant and control tissue in the dorsal hinge. The number of EdU-positive cells in each genotype was then divided by the area (pixels sq) examined (Fig. 4G–H). We reasoned in that if Stat92E mutant cells and Minute/+ cells were undergoing S phase at the same rate, there would be a similar number of EdU positive cells/pixels sq in each genotype. However, we observed a statistically significant decrease (P<0.002) in the number of EdU-positive cells/pixels sq in Stat92E clones (Fig. 4H, magenta bar) as compared to adjacent heterozygous tissue (Fig. 4H, green bar). These data indicate that dorsal hinge cells lacking Stat92E cycle more slowly than control cells.

Reducing Wg signaling dominantly inhibits Stat92E activity in the wing hinge

Prior studies have shown that inhibition of Wg signaling results in reduction of the gap domain and collapse of the two Wg rings onto each other (Zirin and Mann, 2007). Since this phenotype is also observed in Stat92E clones, we wanted to assess if alterations in Wg signaling could affect Stat92E activity. We asked if reduction in Wg signaling - by mis-expression of a dominant-negative TCF (TCF^DN)(van de Wetering et al., 1997) - affected JAK/STAT signaling. Clones over-expressing TCF^DN were small due to reduced growth and survival, consistent with a previous report (Johnston and Sanders, 2003). When these clones resided in the 10xSTAT-GFP endogenous domain in the dorsal hinge, the expression of the Stat92E transcriptional reporter was cell autonomously reduced and the epithelial fold was perturbed within the clone (Fig. 5A–A″), presumably as a result of the decreased survival of TCF^DN-expressing clones. These data suggest that JAK/STAT signaling is genetically downstream of Wg signaling in the wing hinge.

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Figure 5

Wg signaling is dominant to JAK/STAT signaling in the wing hinge

(A) TCF^DN-expressing clones, which inhibit Wg signaling, (marked by βGal (red)) exhibit cell-autonomous loss of 10xSTAT-GFP (green) and the hinge epithelial fold (B′, B″, arrows). Wg is blue. B′ is a close of the area outlined in yellow in B. B″ is single channel for 10xSTAT-GFP.

(B) The wg^spd-fg-LacZ reporter (magenta), corresponding to wg expression in the Wg IR (and at the dorso-ventral boundary), shifts proximally in Stat92E clones, which lack GFP. Red and green arrows in C′, C″ indicate in proximal movement of the wg^spd-fg-LacZ reporter within Stat92E clone boundaries.

(C, D) 10xSTAT-GFP (green) activity is reduced in a third instar wg^spd-fg mutant disc, particularly the distal most fold of reporter expression (C′, D′, arrow). The Wg IR (red) is also lost in this mutant background (C″ D″). C′, D′ is single channel for 10xSTAT-GFP; C″, D″ for Wg.

(E–L) In a second instar (E) and third instar (G) disc from +/SM5~TM6B disc (labeled WT), 10xSTAT-GFP (green) is expressed in two folds in the hinge, one proximal and one distal. Nub is Red and Tsh is blue. F, H show xz scans through the region indicated by the white lines in E and G. Stat92E activity (F″, H″) is concentrated in two domain between the Nub (F″, H″) and Tsh (F, H) domains. In a second instar wg^spd-fg mutant disc, Stat92E activity (green) (I, I′) appears similar to sibling controls (E, E′). However, by mid-third instar, the distal domain of 10xSTAT-GFP expression is greatly reduced in wg^spd-fg (K, K′) compared to sibling controls (G, G′). J, L show xz scans through the region indicated by the white lines in I and K. In a second instar wg^spd-fg disc, Stat92E activity (J″) is concentrated in two domain between the Nub (F″) and Tsh (F) domains similar to sibling controls (F″). However, in a third instar wg^spd-fg disc, Stat92E activity (L″) is greatly reduced in the distal fold (L″, arrow), in stark contrast to a sibling control (H″). F, H, J, L is the merge where 10xSTAT-GFP is green, Nub is red and Tsh is blue. F′, H′, J′, L′ are single channels for 10xSTAT-GFP; F″, H″, J″, L″ for Nub and F, H, J, L for Tsh.

To further investigate the relationship between the Wg and JAK/STAT pathways in the hinge, we examined expression of a wg regulatory enhancer driving expression of lacZ that recapitulates the pattern of the Wg IR wg^spd-fg-lacZ (Neumann and Cohen, 1996). We observed that wg^spd-fg-lacZ moves proximally in Stat92E clones (Fig. 5B′, B″, arrows) in a manner reminiscent of wg-lacZ (Fig. 4G″) or Wg protein (Fig. 4D′, E′). The effect of loss of Stat92E on the wg^spd-fg-lacZ reporter was observed throughout the entire circumference of the Wg IR, as with Wg protein (Fig. 5B″, green arrows). Finally, we examined the expression of 10xSTAT-GFP in the wg^spd-fg mutant. These mutants have reduced expression of the Wg IR, impaired growth of the presumptive hinge and lack distal hinge structures in the adult (del Alamo Rodriguez et al., 2002; Dichtel-Danjoy et al., 2009; Neumann and Cohen, 1996). We carried out these experiments using a stock in which the wg^spd-fg allele was in trans to the SM5~TM6B compound chromosome, which facilitates the identification of homozygous larvae by the lack of Tb. We confirmed that the Wg IR is absent in these discs (Fig. 5C″, D″, arrows). In early to mid-third instar, we find that 10xSTAT-GFP is expressed normally in the dorsal hinge of wg^spd-fg mutants in the two epithelial folds, one proximal and one distal (Fig. 5I–J), as it is in sibling controls (Fig. 5E–F). By late third instar, we find a reduction in the dorsal hinge folds in wg^spd-fg mutants as well as in 10xSTAT-GFP activity in the dorsal and lateral hinge (Fig. 5C′, D′, K′, arrows, Fig. 5K–L). The reduction in the gap domain is particular apparent for the distal fold of 10xSTAT-GFP expression (Fig. 5L″, arrow). By contrast, a third instar disc from a sibling control retained both the proximal and distal folds of 10xSTAT-GFP activity (Fig. 5G–H). These data indicate that the reduction in wg expression in the hinge reduces growth of cells in the gap domain even though they are capable of activating JAK/STAT signaling. Taken together, our results reveal that Wg signaling is dominant to Stat92E activity in these cells. Our results also reveal that JAK/STAT signaling likely does not directly regulate wg expression in the hinge but keeps the Wg IR and OR separated by promoting growth of the gap domain.

No mutual inhibition between Tsh and Stat92E

We analyzed the expression of 10xSTAT-GFP in loss- and gain-of-function clones for Tsh. Since Stat92E activity and Tsh are co-localized in some cells in the proximal hinge, we predicted that Stat92E activity would not be substantially altered when Tsh levels were experimentally manipulated. However, we found that 10xSTAT-GFP is repressed in a cell-autonomous manner when the Tsh-expressing clone arose within the proximal hinge (Fig. 6A′-A, green arrow) but not those that arose in the ventral hinge (Fig. 6A′-A, yellow arrow), despite the fact that Msh is repressed in both clones (Fig. 6A, green and yellow arrow). By contrast, when Tsh-expressing clones arose in the presumptive pouch, we observed induction of this reporter (Fig. 6A′-A, red arrows), presumably as a result of the re-patterning caused by ectopic Tsh (Azpiazu and Morata, 2000; Wu and Cohen, 2002; Zirin and Mann, 2007). We also observed cell-autonomous induction of an upd-lacZ enhancer trap upd^PD1 (Sun et al., 1995) in Tsh-expressing clones in the hinge and in the pouch (Fig. 6B–C, arrows). We reasoned that the ectopic induction of upd by Tsh likely led to autocrine and paracrine activation of Stat92E observed in some Tsh-expressing clones.

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Figure 6

Stat92E and Tsh do not exhibit mutual antagonism

(A) 10xSTAT-GFP is repressed in Tsh-clones residing in the proximal hinge (green arrow, A′, A″) but not in the lateral hinge (A′, A″, yellow arrow), despite Msh being repressed in both clones (A, green and yellow arrow). By contrast, 10xSTAT-GFP is induced in Tsh-expressing clones residing in the pouch (arrows in A′, A). Msh (red) is weakly induced in some Tsh-expressing clones in the pouch (red arrows in A). A′ is single channel for 10xSTAT-GFP; A″ for βGal; A for Msh.

(B, C) An upd enhancer trap (upd^PD1, marked by βGal (red)) is cell-autonomously induced in Tsh-expressing clones (arrows in B–B and C–C), which express both GFP (green) and Tsh (blue). The upregulation of upd occurs in both the hinge (B) and the pouch (B). Note that the endogenous domain of Tsh is not visible in B because the gain of the confocal was decreased to not over-saturate the signal. B′ and C′ are single channel for βGal; B″ and C″ for GFP; B and C″ for Tsh.

(D) Endogenous 10xSTAT-GFP is not altered in a Tsh RNAi (tshi)-expressing clone (marked by βGal (red)) residing in the proximal hinge (arrow in D′-D). The lack of Tsh protein within the clone (arrow in D) indicates the effective of the knockdown. Tsh is blue in D. D′ is single channel for 10xSTAT-GFP ; D′ for βGal; D for Tsh.

We hypothesized that if Tsh represses JAK/STAT signaling to the gap domain, the lack of Tsh should result in increased activity of the pathway, particularly if the clone arose in the proximal hinge. We were unable to examine the effects of tsh loss-of-function clones on Stat92E activity due to the close proximity of the tsh gene to FRT⁸⁰. Instead, we reduced Tsh expression by ectopically expressing a tsh RNAi construct (tshi) (Zirin and Mann, 2007). Although Tsh protein is undetectable in a tshi clone (Fig. 6D, arrow), JAK/STAT signaling is unaltered in it (Fig. D″, arrow). We acknowledge that it is possible that the lack of repression of 10xSTAT-GFP in tshi clones could be explained by a redundant repression by tip-top, a Tsh paralogue that is expressed in an overlapping pattern to that of tsh (Bessa et al., 2009). We also monitored the expression of Tsh in clones that had ectopic JAK/STAT signaling. Tsh expression is not altered in Hop-expressing cells (Fig 7A, A, green arrows). These data suggest that activated Stat92E does not repress Tsh. These data, taken together with the continuous overlap of Tsh with basal JAK/STAT signaling in the dorsal wing disc throughout larval development (Fig. 1H–M), suggest that Tsh and activated Stat92E do not exhibit strong repressive interactions.

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Figure 7

Nub represses Stat92E activity and upd production

(A) Hop-expressing clones (green) moderately repress Nub (red) (small red arrows in A′ and A″) but do not alter Tsh (blue) (green arrows in A). A′ is single channel for GFP; A″ for Nub; A for Tsh.

(B) Nub-expressing clones (red) residing within the hinge autonomously (B′, arrow) repress 10xSTAT-GFP (green). B′ is single channel for 10xSTAT-GFP.

(C) 10xSTAT-GFP extends distally into the pouch in nub¹ mutant clones, which lack βGal (red). Nub is blue. B″ is single channel for 10xSTAT-GFP and the expansion into the pouch is indicated by the arrows.

(D, E) dome-gal4, UAS-GFP (labeled “dome” (green)) is observed only in the dorsal medial hinge (arrow in C′). dome-gal4, UAS-lacZ (marked by βGal (red) in mosaic nub¹ mutant clones (marked by the absence of GFP) is ectopically expressed in the pouch (E′, E″, arrows). Nub is blue. E′ is single channel for GFP; E″ for βGal.

(F) Clonal mis-expression of Stat92E^FL (green) together with Nub (red) does not restore expression of Zfh2 (blue) and hinge folds to the clone, suggesting that Nub represses Stat92E activity and not Stat92E expression. The clone is most easily visualized by ectopic Nub expression. F′ is single channel for Nub; F″ for Stat92E; F for Zfh2.

(G) 10xSTAT-GFP expression domain (green) is separated from the Rn domain (red) by the Wg IR (blue). G′ shows merge of 10xSTAT-GFP and Rn channels. G″ is single channel for 10xSTAT-GFP; G for Rn.

(H, I) Rn-expressing clones that are marked with βgal (red) residing within the hinge autonomously (H′, I′, arrows) repress 10xSTAT-GFP (green). H′ and I′ are single channels for 10xSTAT-GFP.

(J) 10xSTAT-GFP (green) is downregulated when Rn is mis-expressed by MS1096-Gal4. Nub is red and Tsh is blue. Arrows in J′ mark the perturbations to 10xSTAT-GFP in the dorsal hinge. J′ is single channel for 10xSTAT-GFP; J″ for Nub; J for Tsh.

(K) upd-gal4, UAS-GFP (upd>GFP) is ectopically expressed in pouch cells in nub¹ mutant clones marked by the absence of Nub in red and βgal in blue. K′ is the single channel for upd>gfp with the clone boundary outlined by red line.

(L, M) in situ hybridization reveals the expression of upd within the medial hinge in a wild type second instar wing disc (L). In a dpp>nub second instar disc, upd is repressed within the dpp domain (arrow, M).

(N, O) MS1096-driven over-expression of upd (MS1096>upd) in the pouch and dorsal wing results in ectopic activation of the JAK/STAT pathway in the pouch (N′), and small wings in the adult (O).

Nub and Rn restrict JAK/STAT activity to the presumptive hinge

Given the progressive exclusion of Stat92E activity from the presumptive pouch during wing development and the fact that Nub can act as a direct transcriptional repressor (Neumann and Cohen, 1998), we tested the hypothesis that Nub represses of one or more of the components of the JAK/STAT pathway. Cells that ectopically express Nub always exhibit autonomous loss of JAK/STAT pathway activity regardless of their position along the hinge circumference (n ≥300 Nub-expressing clones examined) (Fig 7B′, arrows and data not shown). Furthermore, in nub¹ clones, Stat92E activity is ectopically observed in the pouch along the entire circumference of the hinge-pouch interface (Fig. 7C′, arrows). To rule out the trivial possibility that ectopic expression of Stat92E activity in nub¹ clones was due to spurious results of the 10xSTAT-GFP reporter, we examined expression of dome, another well-characterized Stat92E target gene that encodes the Upd receptor. In the embryo and eye disc the expression of the dome gene is substantially increased by activation of the JAK/STAT pathway in a positive feedback loop (Bach et al., 2003; Flaherty et al., 2009; Lovegrove et al., 2006). In wild type third instar wing discs, the dome>gfp transcriptional reporter is faintly detected in the dorsal hinge (Fig. 7D, arrow). In nub¹ mutant clones, dome is ectopically expressed in the proximal wing pouch (Fig. 7E–E″, arrow). These results indicate that JAK/STAT signaling is increased within the hinge when Nub levels are reduced. Consistent with the model that Nub inhibits Stat92E activity (and not Stat92E expression), we are unable to restore hinge folds and Zfh2 expression in clones that mis-express both Nub and full-length Stat92E transgenes (Fig. 7F–F, arrow).

We note that the ectopic expression of 10xSTAT-GFP and dome do not extend fully into the presumptive pouch within nub clones (Fig. 7C′, E″, arrows). We reasoned that additional pouch factors like Rn might repress JAK/STAT signaling in clones with reduced Nub levels. Indeed, co-labeling for 10xSTAT-GFP, Wg and Rn revealed that Rn and activated Stat92E are separated by the Wg IR (Fig. 7G–G). Consistent with our hypothesis that pouch factors other than Nub can restrict JAK/STAT signaling, we observe cell autonomous repression of 10xSTAT-GFP in Rn-expressing clones in the lateral and ventral hinge (Fig. 7H′, I′, arrows). 10xSTAT-GFP is downregulated in the dorsal hinge when Rn is mis-expressed using MS1096-Gal4 (Fig. 7J′, arrows). The MS1096-Gal4 driver is initially expressed in the dorsal wing disc, and at later larval stages in the ventral pouch (Capdevila and Guerrero, 1994; Neumann and Cohen, 1996).

We also addressed if Nub could inhibit expression the ligand Upd, which is expressed in a characteristic five-spot pattern in a wild-type wing disc (Fig. 1F′ and (Bach et al., 2007; Mukherjee et al., 2005)). Indeed, upd>GFP reporter is ectopically activated in nub¹ mutant clones throughout the dorsal hinge (where it is usually restricted to two dots) and in the proximal wing pouch in a cell-autonomous manner (Fig. 7K, K′). We observe a similar result in nub¹ clones straddling in the lateral and ventral hinge (data not shown). We then tested the hypothesis that Nub represses upd production. We expressed Nub outside of its endogenous domain and monitored upd mRNA by in situ hybridization. In wild type second instar wing discs, upd is expressed within the central hinge (Fig. 7L), consistent with a previous report (Mukherjee et al., 2005). By contrast, upd is strongly repressed within its endogenous domain by mis-expression of UAS-nub along the A/P axis with dpp-gal4 (Fig. 7M, arrow). These results suggest that Nub represses the upd gene. Taken together with results indicating that Nub also inhibits Stat92E activity, we favor the interpretation that Upd synthesis and Stat92E activity are incompatible with cells that have acquired pouch identity.

Finally, we addressed the issue of whether ectopic JAK/STAT signaling in clones could affect expression of Nub. We observed that Nub protein levels are moderately reduced in a cell-autonomous manner in Hop flip-out clones residing in the pouch (Fig. 7A″, arrows). These data suggest that activated Stat92E can moderately repress Nub.

We thank our colleagues, the Bloomington stock center and the DHSB for flies and antibodies. We are particularly grateful to Sonsoles Campuzano for sending the msh allele and Ara antibody to us so quickly. We are very grateful to the members of the NYU Biology Department and Esteban Mazzoni for their generosity in the aftermath of Superstorm Sandy. We also thank two anonymous reviewers whose comments greatly improved this study. This work was supported by March of Dimes Basil O’Connor Starter Award (5-FY06-579), Breast Cancer Alliance, Inc. Young Investigator Award, and NIH grant R01-GM085075 (all to EAB).