Title: Membrane potential regulates Hedgehog signalling in the <i>Drosophila</i> wing imaginal disc
Abstract: Article25 February 2021Open Access Transparent process Membrane potential regulates Hedgehog signalling in the Drosophila wing imaginal disc Maya Emmons-Bell Maya Emmons-Bell Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA Search for more papers by this author Iswar K Hariharan Corresponding Author Iswar K Hariharan [email protected] orcid.org/0000-0001-6505-0744 Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA Search for more papers by this author Maya Emmons-Bell Maya Emmons-Bell Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA Search for more papers by this author Iswar K Hariharan Corresponding Author Iswar K Hariharan [email protected] orcid.org/0000-0001-6505-0744 Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA Search for more papers by this author Author Information Maya Emmons-Bell1 and Iswar K Hariharan *,1 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA *Corresponding author. Tel: +1 510 643 7438; E-mail: [email protected] EMBO Reports (2021)22:e51861https://doi.org/10.15252/embr.202051861 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract While the membrane potential of cells has been shown to be patterned in some tissues, specific roles for membrane potential in regulating signalling pathways that function during development are still being established. In the Drosophila wing imaginal disc, Hedgehog (Hh) from posterior cells activates a signalling pathway in anterior cells near the boundary which is necessary for boundary maintenance. Here, we show that membrane potential is patterned in the wing disc. Anterior cells near the boundary, where Hh signalling is most active, are more depolarized than posterior cells across the boundary. Elevated expression of the ENaC channel Ripped Pocket (Rpk), observed in these anterior cells, requires Hh. Antagonizing Rpk reduces depolarization and Hh signal transduction. Using genetic and optogenetic manipulations, in both the wing disc and the salivary gland, we show that membrane depolarization promotes membrane localization of Smoothened and augments Hh signalling, independently of Patched. Thus, membrane depolarization and Hh-dependent signalling mutually reinforce each other in cells immediately anterior to the compartment boundary. Synopsis Membrane depolarization can promote signaling via the Hedgehog pathway in the Drosophila wing disc. Membrane potential is patterned in the Drosophila wing disc. Cells that express Hedgehog target genes are more depolarized. Membrane depolarization promotes Hedgehog signaling by increasing membrane localization of Smoothened. Introduction During the development of multicellular organisms, cell–cell interactions have an important role in regulating cell proliferation and cell fate specification. In both invertebrates and vertebrates, the Hedgehog (Hh) signalling pathway has been implicated in patterning a number of tissues during development (Lee et al, 2016). Alterations of this pathway have been implicated in human diseases. Reduced Hh signalling can result in congenital abnormalities such as holoprosencephaly (Xavier et al, 2016), and increased activity of the pathway has been implicated in multiple types of cancer (Wu et al, 2017; Raleigh & Reiter, 2019). In recent years, this pathway has been targeted pharmacologically with the goal of reducing its activity in cancers where the pathway is excessively active. Identifying all the ways that this conserved pathway is regulated is of importance in understanding its role in regulating development and for devising ways to alter its activity in disease states. Initially discovered for its role in regulating segment polarity in Drosophila (reviewed in Ingham, 2018), Hh signalling has since been implicated in a multitude of developmental processes. Among the best characterized is the signalling between two populations of cells that make up the Drosophila wing imaginal disc, the larval primordium of the adult wing and thorax. The wing disc consists of two compartments of lineage-restricted cells separated by a smooth boundary. Posterior (P) cells make the morphogen Hedgehog, which binds to its receptor Patched (Ptc), which is expressed exclusively in anterior (A) cells. Hh has a relatively short range either because of its limited diffusion (Tabata & Kornberg, 1994; Strigini & Cohen, 1997), or because it is taken up by nearby target cells via filopodia-like protrusions known as cytonemes (González-Méndez et al, 2017). Hh alleviates the repressive effect of Ptc on the seven-transmembrane protein Smoothened (Smo) in A cells near the boundary, initiating a signalling cascade that culminates in the stabilization of the activator form of the transcription factor Cubitus interruptus (Ci), and expression of target genes such as the long-range morphogen Dpp (Jiang & Hui, 2008; Lee et al, 2016; Petrov et al, 2017). In turn, Dpp regulates imaginal disc patterning and growth in both compartments (Hamaratoglu et al, 2014). While the role of cell–cell interactions, diffusible morphogens and even mechanical forces have been studied in regulating the growth and patterning of the wing disc, relatively little attention has been paid to another cellular parameter, membrane potential or Vmem. Vmem is determined by the relative concentrations of different species of ions across the cell membrane, as well as the permeability of the membrane to each of these ions. These parameters are influenced by the abundance and permeability of ion channels, the activity of pumps, and gap junctions. While changes in Vmem have been studied most extensively in excitable cells, there is increasing evidence that the Vmem of all cells, including epithelial cells, can vary depending on cell-cycle status and differentiation status (Blackiston et al, 2009; Sundelacruz et al, 2009). Mutations in genes encoding ion channels in humans ("channelopathies") can result in congenital malformations (Plaster et al, 2001). Similarly, experimental manipulation of ion channel permeability can cause developmental abnormalities in mice as well as in Drosophila (Dahal et al, 2017; Belus et al, 2018). Only more recently has evidence emerged that Vmem can be patterned during normal development. Using fluorescent reporters of membrane potential, it has been shown that specific cells during Xenopus gastrulation and Drosophila oogenesis appear more depolarized than neighbouring cells (Krüger & Bohrmann, 2015; Pai et al, 2015). A recent study established that cells in the vertebrate limb mesenchyme become more depolarized as they differentiate into chondrocytes, and that this depolarization is essential for the expression of genes necessary for chondrocyte fate (Atsuta et al, 2019). However, in many of these cases, the relationship between changes in Vmem and specific pathways that regulate developmental patterning have not been established. Here we investigate the patterning of Vmem during wing disc development and show that the regulation of Vmem has an important role in regulating Hh signalling. We show that the cells immediately anterior to the compartment boundary, a zone of active Hh signalling, are more depolarized than surrounding cells, and that Hh signalling and depolarized Vmem mutually reinforce each other. This results in an abrupt change in Vmem at the compartment boundary. Results We began by asking whether or not Vmem is patterned in the wing imaginal disc. Wing imaginal discs from third-instar larvae were dissected and incubated in Schneider's medium containing the Vmem reporting dye DiBAC4(3) (hereafter DiBAC) at a concentration of 1.9 µM for 10 min. DiBAC is an anionic, membrane-permeable, fluorescent molecule that accumulates preferentially in cells which are relatively depolarized compared to surrounding cells due to its negative charge and has been used to investigate patterns of endogenous Vmem in non-excitable cells in a variety of organisms (Adams & Levin, 2012; Krüger & Bohrmann, 2015; Atsuta et al, 2019). In contrast to patch-clamp electrophysiology, utilizing DiBAC allowed us to make comparisons of membrane potential across a field of thousands of cells. A stripe of cells running through the middle of the pouch of the wing disc appeared more fluorescent, thus indicating increased DiBAC uptake (Fig 1A and B), suggesting that these cells are more depolarized than surrounding cells. This pattern of fluorescence was observed in more than 35 individual wing imaginal discs and was not observed when imaginal discs were cultured in the voltage-insensitive membrane dye FM4-64 (Fig 1C–E). Patterned DiBAC fluorescence was observed at both apical (Fig 1A–A′) and basal (Fig 1B–B′) focal planes. Addition of the Na+/K+ ATPase inhibitor ouabain to the cultured discs, which depolarizes cells by collapsing transmembrane Na+ and K+ gradients, resulted in increased, and more uniform, DiBAC fluorescence (Fig 1F and G), indicating that patterned DiBAC fluorescence in wing disc tissue was contingent upon mechanisms that normally maintain Vmem. Figure 1. Membrane potential is patterned in the third-instar wing imaginal disc A, B. Live third-instar discs incubated in DiBAC. DiBAC fluorescence is observed in the wing imaginal disc in both apical (A–A′) and basal (B–B′) optical sections. Increased fluorescence is observed in the centre of the pouch (white arrow, B′) and at the dorsoventral (D-V) compartment boundary in the anterior compartment (yellow arrow, (B′), and inset, (D)). C–E. Comparison of DiBAC with the voltage-insensitive dye FM4-64. Incubation of live discs in FM4-64 (E) shows more uniform fluorescence when compared to DiBAC (D). Quantitative comparison of fluorescence in the two white boxes in each panel is shown in (C). N = 7 discs for each treatment, data compared using an unpaired t-test, ** indicates P < 0.001, error bars are standard deviations. The region boxed in yellow in (D) is shown at higher magnification to show DiBAC fluorescence at the D-V compartment boundary. F, G. Incubation in ouabain results in brighter and more uniform DiBAC fluorescence. H–H″. Live wing discs expressing UAS-RFP anterior to the compartment boundary, under the control of ptc-Gal4, were incubated in DiBAC, showing that the stripe of increased DiBAC fluorescence coincides with the posterior edge of ptc expression. White arrowhead in (H) indicates the stripe in the wing pouch; yellow arrowheads indicate the stripe in the dorsal and ventral hinge. I, I′. Early L3 discs incubated in DiBAC. Patterned depolarization is evident throughout the third larval instar, with the stripe of increased fluorescence becoming narrower in more mature discs with developmental time (Compare I with A, bracket in I indicates width of increased DiBAC fluorescence). Data information: Scale bars are 100 µm in all panels, except for (A′ and B′), where scale bars are 50 µm. Download figure Download PowerPoint The position of the stripe of altered Vmem is reminiscent of the anteroposterior (A-P) compartment boundary in the wing disc, which separates two lineage-restricted populations—the A cells and the P cells. In order to identify the population of depolarized cells with respect to the compartment boundary, we examined discs expressing UAS-RFP under the control of patched-Gal4 (ptc>RFP) that had been incubated in DiBAC. ptc>RFP is expressed in those cells that express the highest levels of endogenous ptc, which are immediately anterior to the compartment boundary. The cells in which DiBAC accumulated at higher levels correlated with expression of RFP throughout the third larval instar (Fig 1H–H″), indicating that cells anterior to the compartment boundary are more depolarized than cells across the compartment boundary in the posterior compartment. ptc-expressing cells in the hinge also were more fluorescent upon DiBAC staining (Fig 1H, yellow arrowheads), but for the purposes of this work we focused on the wing pouch (Fig 1H, white arrowhead). As with ptc-Gal4 expression, the domain of relative depolarization was broader in early third-instar wing discs, becoming more and more restricted to cells just anterior to the compartment boundary over the course of developmental time (Fig 1I–I′). Expression and function of endogenous ion channels anterior to the compartment boundary The resting potential, Vmem, results from the activity of a large number of different transporters of charged molecules, as well as the permeability of the membrane to each of those molecules. Thus, the relative depolarization of the region immediately anterior to the compartment boundary is unlikely to result simply from a change in the activity of a single pump or channel. However, by identifying transporters expressed in this region, it should be possible to manipulate Vmem by altering their expression or properties. To that end, we examined a published transcriptome data set (Willsey et al, 2016), comparing the abundance of transcripts in ptc-expressing cells with those of cells in the posterior compartment. In this data set, we noticed several ion channels with differential expression between the two populations of cells. Among these are two members of the Degenerin Epithelial Na+ Channel (DEG/ENaC) family of channels, ripped pocket (rpk) (Adams et al, 1998), and pickpocket 29 (ppk 29) (Thistle et al, 2012). DEG/ENaC channels are members of a diverse family of amiloride-sensitive cation channels. An antibody that recognizes the Rpk protein has been characterized previously (Hunter et al, 2014), which allowed us to examine its pattern of expression. In late L3 wing discs, we found that Rpk was indeed expressed anterior to the compartment boundary, in a stripe of cells that also express ptc>RFP (Fig 2A–A′). In addition, we observed expression of Rpk in cells near the dorsoventral (D-V) boundary. Expression was most obvious in two rows of cells flanking the D-V boundary in the anterior compartment, which are likely to be the two rows of cells arrested in the G2 phase of the cell-cycle (Johnston & Edgar, 1998). Indeed, the pattern of DiBAC uptake in this portion of the wing disc also suggests a very thin stripe of low-fluorescence flanked by two regions of higher fluorescence (see inset in Fig 1D). This is consistent with previous work showing that cells in culture become more depolarized as they progress through S-phase and peaks at the onset of mitosis (Cone, 1969), reviewed in (Blackiston et al, 2009). Thus, at least in principle, the increased expression of Rpk could contribute to the depolarization observed in these regions of the wing disc. Figure 2. Expression of endogenous channels is patterned and contributes to depolarization anterior to the compartment boundary A–A″. Expression of Rpk is increased anterior to the A-P compartment boundary and in two rows of cells flanking the D-V compartment boundary in the A compartment. B–B″. Blockade of DEG/ENaC channels by incubation in amiloride abolishes the stripe of increased DiBAC fluorescence along the A-P compartment boundary (white arrowhead). White boxes indicate regions used to calculate the average DiBAC fluorescence intensity ratio across the compartment boundary = 0.98, standard deviation = 0.05, n = 5 discs. Increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead). The same vehicle was used as in experiments with ouabain, and the control is in Fig 1F. C, C′. Amiloride incubation does not diminish Rpk expression. White arrowhead indicates approximate position of the A-P compartment boundary, yellow arrowhead indicates approximate position of the D-V compartment boundary. D, E. Expression of rpk-RNAi using dpp-Gal4 results in diminished DiBAC fluorescence along the A-P compartment boundary (white arrowhead), while increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead). Data information: Scale bars are 100 µm, except in (B″), where scale bar is 50 µm. Download figure Download PowerPoint Since Rpk, and possibly Ppk29, are expressed anterior to the compartment boundary, we tested the effect of blocking these channels by treating discs with amiloride. Amiloride is predicted to reduce the permeability of DEG/ENaC family channels (Garty, 1994). Addition of amiloride did not alter the pattern of Rpk protein expression (Fig 2C–C′). However, amiloride addition abolished the stripe of increased DiBAC fluorescence anterior to the compartment boundary (Fig 2B–B″). These findings suggest that a conductance mediated by one or more channels of the DEG/ENaC family contributes to the relative depolarization of this region. Amiloride addition did not, however, seem to affect the increased DiBAC fluorescence observed at the D-V boundary (Fig 2B″). Since amiloride likely targets multiple ENaC channels, we also depleted Rpk using an RNAi transgene expressed in cells anterior to the compartment boundary using dpp-Gal4 and Gal80TS, allowing us to express RNAi in only the 48 h prior to dissection. In these discs, we no longer observed the stripe of increased DiBAC fluorescence that ran along the A-P compartment boundary (Fig 2D and E white arrowhead), while increased fluorescence at the D/V boundary was preserved (Fig 2E, yellow arrowhead). Additionally, we knocked down rpk in the wing pouch during the last 48h of larval development using rotund-Gal4 and Gal80TS. rpk knockdown reduced anti-Rpk antibody staining (Fig EV1B and D), validating the efficacy of the dsRNA, and it also reduced DiBAC staining in the wing pouch (Fig EV1A and C). From these experiments, we conclude that reducing either the expression or permeability of endogenous DEG/ENaC family channels, notably Rpk, can abolish the region of depolarization anterior to the A-P compartment boundary. Hence, Rpk, and possibly other DEG/ENaC channels, contribute to this local alteration in Vmem. Click here to expand this figure. Figure EV1. rpk knockdown decreases DiBAC staining and Rpk expression at the A-P compartment boundary A. Expression of rpk-RNAi in the wing pouch decreases patterned DiBAC staining (white arrowhead). B. Expression of rpk-RNAi in the wing pouch decreases anti-Rpk staining anterior to the A-P compartment boundary (white arrowhead). C, D. Control discs. Increased DiBAC staining and anti-Rpk staining are visible in the centre of the wing pouch (white arrowheads). Data information: Scale bars are 100 µm. Download figure Download PowerPoint In most cells, the Na+/K+ ATPase is primarily responsible for setting a negative Vmem, since it uses ATP hydrolysis to extrude three Na+ ions and bring in two K+ ions per cycle of activity (Morth et al, 2007). RNA of ATPα (Lebovitz et al, 1989), which encodes a subunit of the Na+/K+ ATPase, was also detected at higher levels in ptc-expressing cells (Willsey et al, 2016). Using an antibody to ATPα (Roy et al, 2013), we once again observed elevated expression anterior to the compartment boundary, with a hint of increased expression at the D-V boundary (Fig 3A‴). While it is difficult to predict the contribution of patterned expression of each channel to the patterning of Vmem, detection of these channels at the anteroposterior compartment boundary of the wing disc allowed us to manipulate their expression or to pharmacologically alter their properties. Figure 3. Patterned Rpk and ATPα expression and membrane depolarization require Hh signalling A–A‴. Immunostaining of discs expressing ptc>RFP with antibodies to Rpk and ATPα showing elevated levels of both proteins anterior to the compartment boundary. White arrowheads indicate approximate position of the A-P compartment boundary. B–C′. L3 discs that are temperature-sensitive for hh following shift to the restrictive temperature for 12 h show loss of increased expression of Rpk (B, B′) and ATPα (C, C′) anterior to the compartment boundary. Controls shown in Fig EV2. White arrowheads indicate approximate position of the A-P compartment boundary. D–E‴. Clones of cells expressing an activated form of the transcription factor Ci show elevated Rpk and ATPα expression in the anterior compartment (white box) as well as the posterior compartment (yellow arrowhead). (E–E‴) A single clone in the A compartment is shown at higher magnification. F–F′. A clone of cells in the anterior compartment expressing ci3M following incubation in DiBAC. G–G′. A clone of cells in the anterior compartment expressing ptc-RNAi following incubation in DiBAC. The clones also express RFP. Data information: All scale bars are 100 µm, except for (B′), (C′) and (F–G′) where scale bars are 50 µm and (E–E‴) where scale bars are 25 µm. Download figure Download PowerPoint Patterned expression of rpk and ATPα is regulated by Hedgehog signalling Since the increased expression of Rpk and ATPα anterior to the compartment boundary occurs precisely within the region of increased Hh signalling (Fig 3A–A‴), we tested whether manipulating components of Hh signalling pathway could alter expression of Rpk or ATPα. We used a temperature-sensitive hh allele (hhTS2) (Ma et al, 1993) in order to decrease Hh signalling for a short period of time. Larvae were raised at a permissive temperature (18°C) to permit normal hh function during early development and then shifted to a restrictive temperature (30°C) during the third larval instar in order to reduce hh function and dissected 12 h after the temperature shift. Under these conditions, increased expression of either ATPα or Rpk was not observed anterior to the compartment boundary (Fig 3B–C′, controls in Fig EV2A–A″), indicating that a normal level of hh activity is necessary for the increased expression of these proteins anterior to the compartment boundary. Interestingly, some expression of Rpk is still visible near the D-V boundary in the anterior compartment which is likely hh-independent. We then examined the effects of increasing Hh signalling. Since Hh-dependent gene expression in this region typically results from stabilization of the activator form of Cubitus interruptus (Ci) (Aza-Blanc et al, 1997), we generated clones of cells expressing a constitutively active version of Ci (ci3m (Price & Kalderon, 1999)). The ci3m allele has S to A mutations at PKA-phosphorylation sites 1–3, rendering the protein more resistant to proteolytic cleavage. These clones had modest increases in expression of both of Rpk and ATPα (Fig 3D–E‴). Additionally, these clones showed increased DiBAC fluorescence (Fig 3F–F′), as did clones expressing RNAi against ptc (Fig 3G–G′). ci3m-expressing clones showed increased DiBAC fluorescence in both the anterior and posterior compartments (Fig EV3A and A′), consistent with the ability of constitutively active Ci to activate Hh target genes in both compartments. Clones expressing ptc-RNAi only showed increased DiBAC fluorescence in the anterior compartment, as ptc is not expressed in the posterior compartment (Fig EV3B and B′). There was not always perfect concordance of increased DiBAC staining with clones, so to describe this variability we quantified the ratio of DiBAC staining in ci3m-expressing clones to DiBAC staining to control tissue (Fig EV3C). Thus, Hh signalling appears to promote expression of both Rpk and ATPα, as well as relative depolarization of Vmem. Click here to expand this figure. Figure EV2. Patterned Rpk and ATPα expression and membrane depolarization require Hh signalling A–A″. Immunostaining of discs heterozygous for the hhAC allele and upshifted to 30°C for 12 h with antibodies to Rpk and ATPα showing elevated levels of both proteins anterior to the A-P compartment boundary. All scale bars are 100 µm. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Hh pathway activation results in depolarization A–A′. Clones of cells expressing ci3m accumulate more DiBAC relative to surrounding tissue in both the anterior (white arrow) and posterior (yellow arrow) compartments. B–B′. Clones of cells expressing ptc-RNAi accumulate more DiBAC relative to surrounding tissue in the anterior compartment (white arrow), but not the posterior compartment (yellow arrow). C. The ratio of DiBAC fluorescence in ci3m-expressing clones to equally-sized regions of tissue not expressing ci3m. N = 10 clones from three imaginal discs. Individual data points are shown, as well as a box plot showing descriptive statistics. Bounds of the box show lower and upper quartiles, bar within the box shows the median of the data, and whiskers show minimum and maximum bounds of data. Data information: All scale bars are 100 µm. Download figure Download PowerPoint Manipulating expression of endogenous ion channels modulates Hedgehog signalling We then investigated whether a depolarized Vmem is required for the high levels of Hh signal transduction that occur immediately anterior to the compartment boundary. To that end, we reduced rpk expression in the dorsal compartment of the wing imaginal disc using ap-Gal4 (Fig EV4A) and UAS-rpkRNAi. The Hh signal transducer smo is transcribed throughout the Drosophila wing imaginal disc, but immunostaining for Smo protein reveals that it is most abundant on cell membranes in the posterior compartment, and in cells directly anterior to the compartment boundary (Fig 4A–A‴). Membrane localization of Smo in the anterior compartment is thought to depend on high levels of Hh signalling (Zhu et al, 2003). The Hh signalling pathway is active in several rows of cells immediately anterior to the compartment boundary that receive Hh. Additionally, several components of the Hh signalling pathway are active in the entire posterior compartment, possibly because the absence of Ptc renders Smo constitutively active (Ramírez-Weber et al, 2000). However, target genes are not activated in posterior cells because Ci is only expressed in the anterior compartment. Knockdown of rpk resulted in a reduction in membrane staining of Smo in the dorsal compartment, as compared to ventral cells (Fig 4B′). This was observed in both posterior cells which do not express ptc and in anterior cells which do. Thus, at least in posterior cells, the effect on Smo localization does not require ptc function. In anterior cells near the compartment boundary, Hh signalling also results in stabilization of the activator form of Ci. In ap>rpkRNAi discs, the level of activated Ci in the dorsal compartment was reduced (Fig 4B″). We also examined the expression of ptc, which is a direct transcriptional target of Ci (Alexandre et al, 1996). The stripe of staining with anti-Ptc was much fainter in the dorsal part of the disc (Fig 4C). Thus, expression of rpkRNAi reduces Hh signalling in cells anterior to the compartment boundary. Knockdown of ATPα using ap>ATPαRNAi resulted in severely altered tissue morphology (Fig EV4C and D), and a reduction in anti-Ptc staining (Fig EV4D), suggesting that expression of ATPαRNAi also reduces Hh signalling. We have shown earlier that antagonizing the function of the Na/K-ATPase, of which ATPα is a component, with ouabain completely abolishes the patterned depolarization in this disc (Fig 1G). Click here to expand this figure. Figure EV4. Manipulating levels of the ion channel Rpk impacts both Hh and Wg signal transduction A, B. Expression patterns of ap-Gal4 (A) and rn-Gal4 (B). C, D. Immunostaining of Smo protein (red), full-length Ci (light blue) (C–C″) and Ptc protein (D) in discs expressing ATPαRNAi in the dorsal compartment. Brackets indicate tissue expressing RNAi and control tissue. E–F″. Immunostaining of Smo protein (red) and full-length Ci (light blue) in (E–E″) control discs and (F–F″) discs expressing rpkRNAi for 48 h before dissection. G–H′. Immunostaining of Ptc protein in (G, G′) control discs and (H, H′) discs expressing rpkRNAi for 48 h before dissection. I–J″. Immunostaining for Wg protein in (I–I″) control discs and (J–J″) discs expressing rpkRNAi for 48 h before dissection. K–L′. Immunostaining for Cut protein in (K, K′) control discs and (L, L′), discs expressing rpkRNAi for 48 h before dissection. Data information: All scale bars are 100 µm except (D), where scale bar is 50 µm. Download figure Download PowerPoint Figure 4. The ENaC channel Rpk is required for Hh signal transduction A–A‴. Immunostaining of Smo protein (red) and full-length Ci (light blue) in discs expressing en-Venus. Smo is expressed at higher levels in the P compartment and in a stripe 5–10 cells wide just anterior to the A-P compartment boundary. White arrowhead indicates cells just anterior to the A-P compartment boundary. B, C. Effect of expressing an RNAi against the ENaC channel rpk in the dorsal compartment of the disc using ap-Gal4. Knockdown of Rpk in the dorsal compartment results in decreased accumulation of the Hh signal transducer Smo (B, B′) and the activator form of Ci (B, B″). The expression of ptc, visualized using an anti-Ptc antibody (C), a downstream target gene of Hh signalling, is also diminished. The rpk-RNAi line BL:39053 is used in all panels shown, but the phenotype was validated with a second RNAi line (BL:25847). Data information: All scale bars are 50 µm, except for (A) and (B), where scale bars are 100 µm. Download figure Download PowerPoint To examine the effect of altering rpk expression on pathways o