Membrane potential regulates Hedgehog signalling in the Drosophila wing imaginal disc

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.


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 (Gonz alez-M endez et al, 2017). Hh alleviates the repressive effect of Ptc on the seventransmembrane 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 V mem . V mem 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 V mem have been studied most extensively in excitable cells, there is increasing evidence that the V mem 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 V mem 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 (Kr€ uger & 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 V mem and specific pathways that regulate developmental patterning have not been established.
Here we investigate the patterning of V mem during wing disc development and show that the regulation of V mem 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 V mem mutually reinforce each other. This results in an abrupt change in V mem at the compartment boundary.

Results
We began by asking whether or not V mem is patterned in the wing imaginal disc. Wing imaginal discs from third-instar larvae were dissected and incubated in Schneider's medium containing the V mem reporting dye DiBAC 4 (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 V mem in non-excitable cells in a variety of organisms (Adams & Levin, 2012;Kr€ uger & 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 voltageinsensitive membrane dye FM4-64 (Fig 1C-E). Patterned DiBAC fluorescence was observed at both apical ( Fig 1A-A 0 ) and basal ( Fig 1B-B 0 ) 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 V mem .
The position of the stripe of altered V mem 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 0 ).

Expression and function of endogenous ion channels anterior to the compartment boundary
The resting potential, V mem , 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 ▸ Figure 1. Membrane potential is patterned in the third-instar wing imaginal disc. manipulate V mem 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 0 ). 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. 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 0 ). 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 Gal80 TS , 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 Gal80 TS . 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 V mem .
In most cells, the Na + /K + ATPase is primarily responsible for setting a negative V mem , 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 ATPa (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 ATPa (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 V mem , 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.
Patterned expression of rpk and ATPa is regulated by Hedgehog signalling Since the increased expression of Rpk and ATPa 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 ATPa. We used a temperature-sensitive hh allele (hh TS2 ) (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 ATPa or Rpk was not observed anterior to the compartment boundary ( Fig 3B- 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 Hhdependent 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 ATPa (Fig 3D-E‴). Additionally, these clones showed increased DiBAC fluorescence (Fig 3F-F 0 ), as did clones expressing RNAi against ptc (Fig 3G-G 0 ). ci3m-expressing clones showed increased DiBAC fluorescence in both the anterior and posterior compartments ( Fig EV3A and A 0 ), 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 ▸ 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. posterior compartment (Fig EV3B and B 0 ). 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 ATPa, as well as relative depolarization of V mem .

Manipulating expression of endogenous ion channels modulates Hedgehog signalling
We then investigated whether a depolarized V mem 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 In ap>rpk RNAi 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 rpk RNAi reduces Hh signalling in cells anterior to the compartment boundary. Knockdown of ATPa using ap>ATPa RNAi resulted in severely altered tissue morphology (Fig EV4C and D), and a reduction in anti-Ptc staining (Fig EV4D), suggesting that expression of ATPa RNAi also reduces Hh signalling. We have shown earlier that antagonizing the function of the Na/K-ATPase, of which ATPa is a component, with ouabain completely abolishes the patterned depolarization in this disc (Fig 1G).
To examine the effect of altering rpk expression on pathways other than Hh, we reduced rpk expression in the wing pouch using rotund-Gal4 and Gal80 TS (hereafter rn TS ) (Fig EV4B). Larvae were raised at 18°C and then shifted to 30°C for 48 h before dissection and staining. In these discs, anti-Smo and anti-Ci staining were clearly reduced in the wing pouch (Fig EV4E-F″). Anti-Ptc staining was also clearly reduced (Fig EV4G-H 0 ). All of these observations were consistent with reduced Hh signalling. We also observed a modest reduction in the width of the dorsoventral stripe of Wingless expression in discs expressing rpk-RNAi (Fig EV4I-J″). More obvious was the absence of expression of the Notch and Wingless target gene, cut along the dorsoventral margin (Fig EV4K-L 0 ). Thus, in addition to Hh signalling, altering rpk impacts other pathways as well.

Manipulating V mem regulates Smoothened localization in salivary glands
Cells of the wing imaginal disc are quite small and columnar, posing a challenge for subcellular imaging. Cells of the salivary gland are much larger than those of the wing disc, allowing for easier visualization of subcellular protein localization. Additionally, much work characterizing the regulation of Smo has been carried out in this tissue (Zhu et al, 2003). To directly test whether or not altering V mem can modulate Hh signalling, we used the bacterial sodium channel NaChBac, which can be used to cause membrane depolarization in insect cells by overexpression (Ren et al, 2001;Luan et al, 2006;Nitabach et al, 2006). Expression of NaChBac in the salivary gland using the Gal4 driver line 71B-Gal4 showed a clear increase in membrane-associated staining with anti-Smo (Fig 5A, B and D) as well as increased expression of Ptc (Fig 5E and F). Correspondingly, increased expression of the mammalian potassium channel Kir2.1, which would be predicted to hyperpolarize Drosophila cells (Baines et al, 2001;Hodge, 2009), reduces fluorescence at the cell surface and increases intracellular fluorescence (Fig 5C and D). Thus, sustained alteration in V mem modulates Hh signalling as assessed by Smo localization and Ptc expression.
In order to investigate the short-term consequences of altering V mem , we used channelrhodopsin ChR2, which when exposed to blue light causes membrane depolarization (Nagel et al, 2003;Schroll et al, 2006). In addition to providing a second independent way of depolarizing cells, this approach allowed us to examine short-term changes in Hh signalling that occur in response to depolarization. Dissected, ChR2-expressing salivary glands were either kept in darkness or exposed to activating light for variable intervals of time. Compared to glands kept in the dark, light-exposed glands had higher levels of Smo at the cell membrane (Fig 5G-I), suggesting that even relatively short-term depolarization can facilitate Smo accumulation at the cell surface. A time course showed that an increase in Smo membrane abundance was detectable as early as 10 min and appeared maximal after 25 min of activating light (Fig 5M-Q). A red light-activated depolarizing channelrhodopsin ReaChR (Lin et al, 2013;Inagaki et al, 2014) also elicited a similar effect (Fig EV5A-C). Activation of ChR2 did not alter membrane abundance of the integrin component Mys (Bunch et al, 1992) or the integrin-associated protein Talin (Brown et al, 2002;Fig EV5D-G 0   Wietek et al, 2017), resulted in a reduction of Smo on the plasma membrane as well as in internal compartments (Fig 5J-L). This suggests that short-term hyperpolarization generally reduces Smo levels, perhaps due to the fact that inactivated Smo is targeted for proteasome-mediated degradation following internalization, as has been reported previously (Li et al, 2012).

V mem regulates Smo membrane localization independently of Ptc in the wing disc
Since a chemiosmotic mechanism has been hypothesized to drive Ptc-mediated inhibition of Smo (Myers et al, 2017), we wondered if depolarization might reduce the inhibitory effect of Ptc on Smo. If this were the case, ectopically depolarizing cells of the posterior compartment of the wing disc should have no effect on Smo membrane localization, since Ptc is not expressed in these cells (Hooper & Scott, 1989;Nakano et al, 1989). To test this, we expressed ChR2 in the dorsal compartment of the wing disc and exposed dissected wing discs to 25 min of activating light. Smo membrane abundance was increased in the anterior dorsal compartment of these discs as compared to discs not exposed to light To test the effects of sustained depolarization during development, we expressed the red light-activated optogenetic channel ReaChR, which is activatable through the larval cuticle, in the dorsal compartment of the wing disc. Animals were raised on a 12-h light/dark cycle, and imaginal discs were harvested in the third larval instar and stained with an anti-Smo antibody. Under these conditions, membrane abundance of Smo was markedly increased in the dorsal portion of both anterior and posterior compartments in these discs, as compared to the ventral compartments (Fig 6D and  D 0 ), indicating that depolarization can mediate increased Smo membrane abundance independently of Ptc. The tissue was also overgrown, in line with other reported phenotypes of Hh pathway hyperactivation (Christiansen et al, 2012). In the wing pouch, expression of the Hh target gene ptc was increased in the dorsal compartment of discs expressing ap>ReaChR (Fig 6E and E 0 ). Thus, optogenetic manipulation of V mem in vivo can regulate Smo localization and abundance even in cells that do not express Ptc.

Discussion
In this study, we show that V mem is patterned in a spatiotemporal manner during development of the wing disc of Drosophila and that it regulates Hedgehog signalling at the compartment boundary. First, we have shown that cells immediately anterior to the compartment boundary are relatively more depolarized than cells elsewhere in the wing pouch. This region coincides with the A cells where Hh signalling is most active, as evidenced by upregulation of Ptc. Second, we found that the expression of at least two regulators of V mem , the ENaC channel Rpk and the alpha subunit of the Na + /K + ATPase are expressed at higher levels in this same portion of the disc. Third, by altering Hh signalling, we demonstrate that the expression of both Rpk and ATPa is increased in cells with increased Hh signalling. Fourth, by manipulating Hh signalling in the disc and using optogenetic methods, both in the salivary gland and wing disc, we show that membrane depolarization promotes Hh signalling as assessed by increased membrane localization of Smo, and expression of the target gene ptc. Thus, Hh-induced signalling and membrane depolarization appear to mutually reinforce each other and thus contribute to the mechanisms that maintain the segregation of A and P cells at the compartment boundary.
Developmentally regulated patterning of V mem in the wing disc We have observed two regions of increased DiBAC fluorescence in the wing imaginal disc. We did not observe obvious upregulation of Rpk and ATPa in other discs, and therefore, our studies have focused on the region immediately anterior to the A-P compartment boundary in the wing disc. In the late L3 wing disc, we observed a region of increased DiBAC fluorescence in the A compartment in the vicinity of the D-V boundary. This corresponds to a "zone of non-proliferating cells" (ZNC) (O'Brochta & Bryant, 1985). Interestingly, the ZNC is different in the two compartments. In the A compartment, two rows of cells are arrested in G2 while in the P compartment, a single row of cells is arrested in G1 (Johnston & Edgar, 1998). Our observation of increased DiBAC fluorescence in the DV boundary of only the A compartment is consistent with previous reports that cells become increasingly depolarized as they traverse S-phase and enter G2 (Cone, 1969), reviewed in (Blackiston et al, 2009). In contrast, cells in G1 are thought to be more hyperpolarized. Additionally, we observed increased expression of the ENaC channel Rpk in two rows of cells at the D-V boundary in the anterior compartment (Fig 2A″), indicating that increased expression of Rpk could contribute to the depolarization observed in those cells. We note, however, that the increased DiBAC fluorescence in these cells was not entirely eliminated by exposing discs to amiloride, indicating that other factors are also likely to contribute.
How does membrane depolarization relate to Hh signalling?
Our data are consistent with a model where membrane depolarization and Hh-induced signalling mutually reinforce each other in the cells immediately anterior to the compartment boundary. Both membrane depolarization and the presence of Hh seem necessary for normal levels of activation of the Hh signalling pathway in this region; neither alone is sufficient (Fig 7). First, we have shown that Hh signalling promotes membrane depolarization. We have also shown that the expression of Rpk just anterior to the A-P compartment boundary is dependent upon Hh signalling. Elevated Rpk expression is not observed when a hh ts allele is shifted to the restrictive temperature, and cells become more depolarized when Hh signalling is constitutively activated through expression of the ci3m allele. Previously published microarray data suggest that Rpk as well as another ENaC family channel Ppk29 are both enriched in cells that also express ptc (Willsey et al, 2016). However, there is no antibody to assess Ppk29 expression currently. The sensitivity of the depolarization to amiloride indicates that these and other ENaC channels make an important contribution to the membrane depolarization. Second, we have shown that the depolarization increases Hh signalling. The early stages of Hh signalling are still incompletely understood (reviewed by Petrov et al, 2017). Hh is thought to bind to a complex of proteins that includes Ptc together with either Ihog or Boi. This alleviates an inhibitory effect on Smo, possibly by enabling its access to specific membrane sterols. Interestingly, it has recently been proposed that Ptc might function in its inhibitory capacity by a chemiosmotic mechanism where it functions as a Na + channel (Myers et al, 2017). An early outcome of Smo activation is its localization to the membrane where its C-terminal tail becomes phosphorylated and its ubiquitylation and internalization are prevented (Zhang et al, 2004;Li et al, 2012). By manipulating channel expression in the wing disc, and by optogenetic experiments in both the salivary gland and wing disc, we have shown that membrane depolarization can promote Hh signalling as assessed by increased Smo membrane localization and increased expression of the target gene ptc. The time course of Smo activation is relatively rapid (over minutes) and is therefore unlikely to require new transcription and translation. In the P compartment, membrane Smo levels are elevated likely because of the complete absence of Ptc, and some downstream components of the Hh signalling pathway are known to be activated (Ram ırez-Weber et al, 2000). However, since Ci is not expressed in P cells, target gene expression is not induced. In the cells just anterior to the boundary, the partial inhibition of Ptc by Hh together with membrane depolarization seem to combine to achieve similar levels of Smo membrane localization. More anteriorly, the absence of this mutually reinforcing mechanism appears to result in Smo internalization (Fig 7). Our experiments do not point to a single mechanism by which depolarization promotes Hh signalling. It is possible that depolarization results in increased Ca 2+ levels by opening Ca 2+ channels at the plasma membrane or by promoting release from intracellular sources (e.g. the ER or mitochondria). Indeed, there is evidence that Ca 2+ entry into the primary cilium promotes Hh signalling, and recent work shows that targets of Sonic Hedgehog (Shh) signalling during mammalian development is augmented by Ca 2+ influx (Delling et al, 2013;Klatt Shaw et al, 2018). A second possibility is that membrane depolarization could, by a variety of mechanisms, activate the kinases that phosphorylate the C-terminal tail of Smo and maintain it at the plasma membrane in an activated state. Depolarization could also impact electrostatic interactions at the membrane that make the localization of Smo at the membrane more favourable. Since Rpk and ATPa are expressed at higher levels in the cells that receive Hh, which have been postulated to make synapse-like projections with cells that produce Hh (Gonz alez-M endez et al, 2017), it is conceivable that these channels could modulate synapse function. Additionally, while our work was under review, it has been reported that reducing glycolysis depletes ATP levels and results in depolarization in the wing imaginal disc, reducing the uptake of Hh pathway inhibitors and stabilizing Smo at the cell membrane (Spannl et al, 2020). Importantly, all these mechanisms are not mutually exclusive and their roles in Hh signalling are avenues for future research.
It is now generally accepted that both cell-cell signalling and mechanical forces have important roles in cell fate specification and morphogenesis. Our work here adds to a growing body of literature suggesting that changes in V mem , a relatively understudied parameter, may also have important roles in development. Integrating such biophysical inputs with information about gene expression and gene regulation will lead to a more holistic understanding of development and morphogenesis. Cells in the posterior compartment (blue) produce the short-range morphogen Hedgehog, which is received by cells immediately anterior to the compartment boundary, in the anterior compartment (dark green). Hh signal releases Ptc-mediated inhibition of Smo, allowing for the stabilization of the transcriptional co-effector Ci, and increased expression of the ENaC channel Rpk, which depolarizes cells immediately anterior to the compartment boundary. Depolarization results in increased membrane abundance of the Hh activator Smo and increased transduction of Hh signal. This, in turn, increases expression of Rpk, reinforcing high levels of Hh signal transduction in the region immediately anterior to the compartment boundary.

Drosophila strains and husbandry
Animals were raised on standard medium as used by the Bloomington Drosophila Stock Center. All animals were raised at 25°C, except ap>rpk-RNAi crosses, which were raised at 18°C to reduce lethality. Crosses using GAL80 TS and the hh TS2 allele were raised at 18°C and then upshifted to 30°C at the reported timepoints.

Live imaging and optogenetics
Larvae were washed with 70% EtOH and PBS prior to dissection. Live tissue was dissected in Schneider's media (#21720001, Gibco), and care was taken to not damage or stretch tissue. For DiBAC staining, imaginal discs were incubated in 1.9 µM DiBAC 4 (3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol; DiBAC 4 (3); Molecular Probes)) in Schneider's media for 10 min with gentle rotation. A small amount media was used to mount the discs, such that the addition of a coverslip did not destroy the tissue, and discs were imaged right away. 100 µM amiloride (#A7419, Sigma-Aldrich) and 100 µM ouabain (#O3125, Sigma-Aldrich) were added to DiBAC solution for pharmacology experiments. Discs imaged in FM4-64 dye (#T13320, Thermo Fisher) were incubated in 9 µM FM4-64, and imaged without washing, to preserve staining of the cell membrane.
For optogenetics experiments in the salivary gland, carcasses were dissected and cleaned (fat body removed) in Schneider's medium. Carcasses were loaded onto a glass cover slip in a large drop of Schneider's, and either kept in the dark (control condition) or exposed to activating light (480 nm for ChR2 and ChloC experiments, 647 nm for ReaChR experiments). After exposure, carcasses were immediately fixed and prepared for immunohistochemistry. For optogenetics experiments using the ChR2 channel in the wing imaginal disc, carcasses were dissected and cleaned (fat body removed) in Schneider's medium, then either fixed (0 min activating light condition), or loaded onto a glass cover slip in a large drop of Schneider's, and exposed to 25 min of activating light (480 nm). Following activation, discs were fixed and prepared for immunohistochemistry. For experiments using the ReaChR channel in the wing imaginal disc, flies were raised on a 12-h light/dark cycle, and imaginal discs were harvested and prepared for immunohistochemistry during the third larval instar.

Mosaic tissue generation
To generate clones expressing ci3m or ptc RNAi , hsFLP;;act<STO-P<UAS-RFP/S-T virgin females were crossed to UAS-Ci3m or UAS-Ptc RNAi males. Larvae were collected as described above, and vials were subjected to a 10 min heat shock in a 37°C water bath 48 h before dissection and live imaging in DiBAC, or fixation and preparation for immunohistochemistry.

Data availability
This study includes no data deposited in external repositories.
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