To begin to characterize the determinants of AWM mechanosensory bristle polarity, we labeled the externally exposed socket and shaft cells in wildtype (wt) and pk^pk mutants with anti-Su(H) (Suppressor of hairless) antibody and phalloidin, respectively. In wildtype control wings at 36 hr apf, the apical ends of socket cells are tilted toward the distal end of the wing and are interspersed with the actin bundles of the shafts (Figure 1C,C’, Figure 1—figure supplement 1B–B’’). The shafts are interspersed between socket cells, and appear to ascend along the proximal side of the adjacent socket cell and pass through an opening at its apical surface. Consistent with polarity patterns of adult bristles, actin bundles of pk^pk mutant shafts near the distal end of the wing show a reversed, proximal, tilt, whereas shafts in the proximal and the very most distal regions show the normal distal tilt (Figure 2A,A’, Figure 1—figure supplement 1C–C’’). Between these regions, shafts show a smooth transition between proximal and distal tilt, with some shafts pointing straight up (neutral tilt). The socket cells tilt at angles that correlate with the polarity of neighboring actin bundles in pk^pk mutant wings: P-D tilt at the proximal region and D-P tilt at the distal region, with smooth transition between those regions (Figure 1—figure supplement 1C–C’’). Furthermore, shaft actin bundles with reversed polarity appear to be positioned on the distal side of the socket cell through which they pass, opposite to their relationship in wildtype and to their relationship in the proximal region in pk^pk mutant wings where their tilt shows the normal, distal, direction (Figure 1—figure supplement 1B–B’’, compare with Figure 1—figure supplement 1C–C’’).

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Previous studies of AWM bristle ultrastructure have been insufficiently detailed to appreciate the determinants of polarity (Hartenstein and Posakony, 1989; Palka et al., 1979). To better understand the structures of shaft-socket pairs, and to unambiguously determine the relationship between sibling shaft-socket cell pairs, random individual bristle lineages were labeled by clonal expression of RFP, filling the cell bodies of labeled shaft and socket cells. Simultaneous staining of the socket cells (identified by Su(H) expression) allows one to identify the sibling shaft-socket cell pairs. 3D confocal reconstructions facilitate examination from a variety of viewpoints and enable the positions and shapes of cell bodies, nuclei, shaft, and apical opening of the socket cells to be visualized (Figure 1B–B’’’, Figure 1—video 1). These views allow us to see that the wildtype nucleus and main portion of the shaft cell is dorsal and extends slightly posterior to that of the sibling socket cell. The base of the shaft rises from the cell body, wraps clockwise along its socket sibling (as viewed from the apical side in a right wing), and then rises through a groove in the socket that extends apically along the proximal side of the socket, finally emerging through the donut shaped apical surface of the socket cell (Figure 1D–D’’, Figure 1—video 1). In contrast, in the distal bristles of pk^pk mutant wings where bristles are reversed, the shaft is positioned within an oppositely oriented groove on the distal side of the sibling socket cell. The shaft nucleus is dorsal and posterior to the socket nucleus, similar to their arrangement in wildtype, though their alignment is not as regular. Therefore, the structure of pk^pk mutant bristles is roughly a mirror image of that in wildtype, with nuclei in similar positions, but the shaft bending in a counterclockwise direction and rising through a groove on the opposite side of the socket cell compared to wildtype (Figure 2B–B’’). In the proximal region of the pk^pk mutant wing, the relationship of shaft-socket siblings resembles that in wildtype, reflecting the normal bristle polarity in that region (Figure 1—figure supplement 1C–C’’ and Figure 3—figure supplement 1B). Therefore, the P or D position of shaft relative to the sibling socket cell correlates to the normal or reversed bristle polarity in proximal and distal regions of pk^pk mutants, suggesting that the shaft position relative to the socket determines bristle polarity (tilt).

Since the relative position of the shaft to the socket appears to be important for bristle polarity, we wished to identify the developmental process by which this relationship is achieved. In microchaete of the notum, the orientation of the initial division of the sensory organ precursor cell is specified by PCP signaling (Schweisguth, 2015). Assuming the orientations of subsequent daughter cell divisions are similarly regulated, the shaft-socket relationship may be determined by their relative positions at their birth. A similar process might occur in AWM mechanosensory bristles. Alternatively, a post-division morphogenetic process may determine their final configurations.

To examine this process, we analyzed 3D structures of shaft-socket sibling pairs at earlier developmental stages. In wildtype bristles at 24 hr apf, the shaft cell nucleus is posterior and just slightly dorsal to the socket cell nucleus, similar to their positions at 36 hr apf (Figure 3A,B). The extending shaft is just reaching the apical surface, and is positioned in a groove on the dorsal side of the socket cell. At the apical surface, the shaft, sits in a shallow indentation in the apical surface of the socket cell, which adopts a crescent shape with the opening of the crescent pointing in the dorsal direction. Over time, from 28 to 32 to 36 hr apf, as the shaft continues to extend above the apical surface, the socket cell crescent rotates clockwise, and gradually closes to form a ring around the shaft (Figure 3A,B, Table 2, Table 1, Figure 3—videos 1, 2, 3). Because the apical surface rotates while the nuclei remain relatively stationary, the shaft and socket twist to form a left-handed helical shape. As the shaft grows out above the socket cell, it points distally. The cell bodies, are initially relatively flat in the dorsal-ventral direction, but as they grow, extend dorsally, becoming flatter in the anterior-posterior direction. Quantification of rotation was performed by measuring rotation angles, as diagrammed in Figure 3B, from 3D images of socket cells captured at different time points (c.f. Figure 3C), and data are displayed in rose plots (Figure 3F, Figure 2—figure supplement 1A, Table 2). Rotation at 32 hr averages 55° in the clockwise direction. Note that throughout, we describe analyses of right wings. In all cases, left wings develop as mirror images of right wings.

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Based on stereotypical patterns from images of timed, fixed samples of the apical surface, we infer that a margin cell at the dorsal side of the shaft-socket pair rotates together with the pair toward the proximal side, generating new junctions with the neighboring socket and margin cells and widening the gap between shaft-socket pairs (Figure 2—figure supplement 1B). During these events, the number of margin cells surrounding the shaft-socket pair is maintained and cell-cell junctions are remodeled.

To characterize the events leading to reversed bristle polarity in pk^pk mutants, rotation angles of shaft-socket pairs during development were analyzed as above. At 24 hr apf, shaft cells were positioned dorsal to their socket sibling cells, as in wildtype. At later times, apical rotation proceeded counterclockwise, opposite to the wildtype direction, in the distal bristles that adopt a reversed polarity, giving rise to a right-handed helical shape in contrast to the left-handed helical shape of wildtype pairs (Figure 3E, Figure 2—figure supplement 1A, Table 3). Shaft-socket pairs in the proximal region rotated clockwise, corresponding to their normal polarity, and bristles in the region between normal and reversed bristles rotated very little, corresponding to their neutral, upright, polarity (Figure 3—figure supplement 1B). Therefore, bristle polarity does not depend on the birth positions of shaft and socket cells, which are born by 14 hr apf (Hartenstein and Posakony, 1989), but rather on the direction of apical rotation of the developing shaft-socket pair, beginning shortly after 24 hr apf.

Since pk^pk mutants show counterclockwise rotation leading to reversed shaft-socket positioning (D-P) and bristle reversal, and Pk^sple overexpression similarly reverses bristle polarity as previously reported (Figure 3—figure supplement 1E,E’ and Gubb et al., 1999), we surmised that Pk^sple induces the counterclockwise helical growth that leads to D-P orientation of shaft-socket pairs and reversed bristle polarity. Consistent with this, counterclockwise rotation of shaft-socket pairs and proximal polarity in pk^pk mutants is suppressed by removing pk^sple (in pk^{pk-sple13/pk-sple13}; Figure 3—figure supplement 1A–D’, Table 3, Gubb et al., 1999), and pk^sple overexpression induces counterclockwise rotation similar to that in pk^pk mutants (Figure 3—figure supplement 1E,E’ Table 2, Table 3). Pk^sple is therefore needed to reverse shaft-socket rotation in pk^pk mutants. Overexpression of Pk^sple induced counterclockwise rotation and reversed polarity in a wider region than in pk^pk mutants (compare Figure 3—figure supplement 1B to E). Thus, endogenous Pk^sple is only poised to act at the distal margin, but exogenous Pk^sple can reverse most, if not all, bristles. Though the potential for Pk^sple to reverse bristle polarity is unmasked in the absence of Pk^pk, it plays no essential role in wildtype polarization, as pk^sple bristles fully rotate, or perhaps marginally over-rotate (59.3°±4.2° vs 54.6°±2.9°, p=0.0702; Table 3, Figure 3—figure supplement 1C,C’).

Surprisingly, pk^pk-sple13 mutant bristles rotate only moderately less than wildtype bristles (47.6°±6.6° vs. 54.6°±2.9°, p=0.0143; Table 3), suggesting that Pk^pk might play only a modest role in controlling the magnitude of rotation in wildtype. We propose that this is due to residual core PCP signaling activity observed in the absence of Pk (Strutt and Strutt, 2007; Lawrence et al., 2004; Adler et al., 2000), and the implications of this result are considered more fully in the Discussion.

We have shown that in wildtype bristles, Pk^pk antagonizes Pk^sple to direct clockwise rotation of shaft-socket pairs, and that Pk^sple, when overexpressed, outcompetes Pk^pk to direct counterclockwise rotation. Although the idea that Pk^pk and Pk^sple antagonize each other has been previously proposed (Gubb et al., 1999), how this occurs has been obscured in part by the inability to specifically visualize the endogenous expression of each isoform. We therefore modified the endogenous genomic sequence encoding Pk^pk or Pk^sple by appending a V5 tag to the N-terminus, facilitating the tissue and cellular level evaluation of their native expression patterns at various developmental stages. Both tagged isoforms support wildtype polarity development in all tissues and various controls suggest that expression of these genomically tagged versions reflect that of the native loci (Figure 4, Figure 4—figure supplements 1 and 2). Here, we describe their expression in developing wings.

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Notably, V5::Pk and V5::Sple reveal that throughout wing development, expression is spatiotemporally dynamic. Early in wing development, Pk^pk protein is strongly expressed and is present in most or all cells. In discs, Pk^pk is relatively elevated in AWM proneural cells. Following a slight dip in levels around 8 hr apf, expression levels climb, peaking around 32 hr apf, when wing hairs emerge, and then decline with little detectable expression remaining by 40 hr apf (Figure 4A–A’’, Figure 4—figure supplement 1B–H,K–K’’). Expression of Pk^sple is below detection in discs (Figure 4—figure supplement 1B,I,J), makes a small peak at around 8 hr apf, and is then not detectable between 16 and 28 hr apf. Beginning around 28 hr apf, Pk^sple expression is specifically detected in dorsal AWM cells and in vein L3. This expression persists through 32 hr apf, when wing hairs emerge, and weaker expression in other veins becomes apparent (Figure 4B–B”, Figure 4—figure supplement 1B,L,L’). At these times, Pk^sple is below detection levels on Western blots. Beginning sometime after 32 hr apf, Pk^sple expression increases in most cells, with its level equaling that of the declining Pk^pk by 36 hr apf and reaching its highest level at around 40 hr apf (the latest time we examined), when Pk^pk is no longer detected. At 40 hr apf, when Pk^sple is at peak expression in most of the wing blade, expression has disappeared from vein cells (Figure 4—figure supplement 1B,L–L”).

Most pertinent to bristle development, at the 24 hr apf AWM, Pk^pk is expressed at apical junctions at similar levels in anterior margin and socket cells (Figure 4C,C’, Figure 4—figure supplement 2A). By 28 hr apf, Pk^pk expression begins to decrease during bristle-socket rotation, first in socket cells, and later in all cells at and near the margin (Figure 4—figure supplement 2A). At the same time, Pk^sple expression becomes evident and increases over time, first uniformly in cells near the margin, and gradually becoming strongest in the socket and shaft cells (Figure 4D,D’,F–F’’, Figure 4—figure supplement 2B, compare with Pk^pk (V5::Pk) in Figure 4E). The timing of the shift from Pk^pk to Pk^sple expression is accelerated at the margin relative to the interior of the wing. Mosaic experiments demonstrate that Pk^pk localizes proximally in socket and other margin cells, and that, importantly, Pk^sple colocalizes with Pk^pk to the proximal side of socket cells. Proximal Pk^sple localization is an unexpected observation based on previous studies in which overexpressed Pk^sple localized at the distal junctions of hair cells (Ayukawa et al., 2014; Ambegaonkar and Irvine, 2015). Because Pk^sple expression is stronger in socket cells compared to margin cells by 32 hr apf, it has the useful property of effectively being expressed as a mosaic, allowing its localization to be scored without inducing clones (Figure 4F–F’’,H–H’’,I–I’’).

As Pk^sple activity reverses rotation direction of shaft-socket pairs in pk^pk mutant wings, Pk^sple protein localization was analyzed in pk^pk mutant wings. Because anti-Pk[C] antibody recognizes the common region of Pk^pk and Pk^sple, the antibody reveals Pk^sple isoform localization in pk^pk mutants. In the region of pk^pk mutant wings where P-D reversal occurs, Pk^sple protein localized at the distal side of socket cells, whereas in the proximal region where polarity is not reversed, Pk^sple protein shows minimal asymmetry (Figure 4G–G’’, Figure 4—figure supplement 2C–C’’’). Distal localization of Pk^sple in the region of polarity reversal was verified by clonal knockdown of pk^sple in pk^pk mutant wings. Notably, the socket-shaft pairs that lacked both Pk^sple and Pk^pk failed to rotate (Figure 4—figure supplement 2D–E’’’).

These results suggest that Pk^pk normally inhibits Pk^sple from localizing distally by recruiting it to the proximal junction of socket cells (and likely also in nearby margin cells, although this is hard to visualize due to lower expression in those cells). Furthermore, the distal localization of Pk^sple in pk^pk mutants correlates with its ability to determine counter-clockwise rotation of shaft-socket pairs on a cell-by-cell basis, suggesting that this localization is likely the determinant of counter-clockwise rotation.

Our results thus far show that the direction of Pk polarization, whether Pk^pk, Pk^sple or both, corresponds to the direction of bristle polarization. Suppression of polarity reversal in pk^pk mutants by dsh implicates the core PCP signaling mechanism in this process Gubb et al. (1999). We therefore asked whether the remaining components of the core PCP signaling mechanism contribute to AWM bristle polarization. As with dsh mutation, knock down of fz or vang in pk^pk mutants suppressed reversal of bristle tilt, shaft-socket orientation, and rotation direction (Figure 5A–D). Core PCP signaling is therefore required for reversed, Pk^sple-dependent polarity.

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To assess a potential contribution of core PCP signaling to Pk^pk-dependent bristle polarization, the anterior region of adult wings from fmi RNAi, fz, vang, and dsh mutants were analyzed, and the rotation of shaft-socket pairs was evaluated for each genotype (Figure 5E–H). Adult mechanosensory bristles of core PCP mutants are less tilted toward the distal direction than those of wildtype and the tilting angles are somewhat irregular, with some bristles tilting out of the plane of the wing. Consistent with the adult wing defects, in pupal wings the shaft position relative to the socket varies, sometimes abruptly, in the same mutant wing, showing less local correlation than in wildtype. Quantification reveals substantial under-rotation of shaft-socket pairs, and a broader distribution of rotation angles than in wild type (Figure 5E–H, compare with Figure 3D,F, Table 2). Thus, careful morphological analysis reveals that core PCP signaling is required for normal, Pk^pk-dependent polarity as well as reversed, Pk^sple-dependent polarity.

Consistent with a role for core PCP components in mediating rotation of shaft-socket pairs, junctional asymmetry of Fz::EGFP and Vang::EYFP is well preserved between socket and margin cells and between adjacent margin cells (Figure 5I–J’). Little accumulation was observed at junctions between shaft and socket cells. Mosaic analyses demonstrated the expected distal localization of Fz and proximal localization of Vang at both margin cell-margin cell and margin cell-socket cell junctions, suggesting that PCP signaling likely occurs between margin cells and between margin and socket cells (Figure 5K,L). Similarly, the reversed bristle polarity observed in pk^pk mutants was accompanied by reversed Vang localization in pk^pk mutant socket and margin cells, consistent with the idea that the direction of core PCP polarization is reversed in pk^pk mutants (Figure 5—figure supplement 1). To functionally test polarity propagation between margin and socket cells by core PCP components, fz or vang knock-down clones were generated, and clones at the AWM were analyzed (Figure 6, Figure 6—figure supplement 1). fz or vang knock-down clones, whether in just margin cells, just shaft-socket pairs, or both, showed non-autonomy as assessed by sequestration of Fz (fz clones) or Vang (vang clones) at the clone borders. Near fz RNAi clones, distal cells, including both bristle and hair cells, were re-oriented: sockets on the distal side of the clones showed counter-clockwise rotation, and hairs on the distal side grew toward the clones. Similarly, sockets on the proximal side of vang RNAi clones rotated counter-clockwise (Figure 6—figure supplement 1). These results indicate that core PCP signaling propagates between bristle and margin cells to control the rotation direction of shaft-socket pairs.

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We have previously proposed that a signal from the Ft/Ds/Fj system provides a directional cue to orient core PCP signaling in some tissues (Ma et al., 2003; Yang et al., 2002; Matis et al., 2014; Olofsson et al., 2014), although others have argued that this system operates in parallel with core PCP signaling (Casal et al., 2006; Lawrence et al., 2007; Brittle et al., 2012). An asymmetry of Ft-Ds heterodimers, with a small excess of Ds displayed on the distal side of the cell, and Ft on the proximal side, has been observed, and is proposed to provide this signal (Ambegaonkar et al., 2012; Bosveld et al., 2012; Brittle et al., 2012). We have also proposed that the core PCP module differentially interprets directional signals from Ds when operating in Pk^pk- or Pk^sple-dependent modes, with Pk^sple directing localization of the Fmi-Vang complex to the side where Ds is in excess, while the Fmi-Vang complex localizes to the opposite side when functioning in a Pk^pk-dependent manner [(Olofsson and Axelrod, 2014); see also Lawrence et al. (2004). Pk^sple has been shown to bind to Ds and the associated Dachs protein, providing a mechanism for orienting Pk^sple-dependent core function to the Ft/Ds/Fj signal (Ayukawa et al., 2014; Ambegaonkar and Irvine, 2015), whereas a less direct, microtubule-dependent mechanism was proposed to mediate this response when Pk^pk is predominant (Shimada et al., 2006; Harumoto et al., 2010; Olofsson and Axelrod, 2014; Matis et al., 2014). In contrast, some have proposed that Pk^pk-dependent core signaling is instead uncoupled from the Ft/Ds/Fj signal (Merkel et al., 2014; Ambegaonkar and Irvine, 2015).

We reasoned that our reagents would allow us to analyze effects of the Ft/Ds/Fj signal on each Pk isoform. We first examined the phenotype resulting from knockdown of Ds (Figure 7C). As previously observed (Adler et al., 1998), normal bristle tilt was substantially disturbed. The pattern of disturbance consistently showed regions of coordinated polarity that smoothly transition through neutral polarity to adjacent regions of opposite polarity, although the number and position of those domains varied. The effectively mosaic expression of V5::Sple localization allows one to observe precisely correlated regions of distal and proximal Sple localization corresponding to the regions of reversed and normal polarity, respectively, with relatively symmetric localization in the intervening transitions (Figure 7C). These results suggest that local core PCP signaling maintains polarity correlation among immediate neighbors but that alignment to the tissue axis is eliminated in the absence of Ds.

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We then asked if the Pk^sple-dependent reversed polarity in pk^pk mutants depends on Ds. When ds was knocked down in pk^pk mutants, bristle reversal was blocked, producing a phenotype similar to that of core mutants, and the distal localization of Pk^sple was no longer observed (Figure 7A–D). Therefore, the reversed, Pk^sple-dependent polarity in pk^pk mutants requires Ds, and we interpret this to indicate that the Ds global signal recruits Pk^sple to sites of enriched Ds (distal) in the absence of Pk^pk (Figure 7B; compare with 7D), which drives reversal of shaft-socket orientation and therefore reversal of bristle polarity.

In ds knockdowns, we are unable to readily interpret the localization pattern of V5::Pk because levels are similar in socket and margin cells. Nonetheless, other results suggest that the local polarity correlation in the absence of Ds is mediated primarily by asymmetric localization of Pk^pk rather than the asymmetric localization of Pk^sple that we can observe. First, recall that in wildtype, Pk^sple is recruited to colocalize with Pk^pk at proximal sites, so Pk^pk is likely to similarly recruit the colocalization of Pk^sple in the absence of Ds. Consistent with this idea, when ds was knocked down in pk^pk mutants, neither proximal nor distal localization of Pk^sple was observed in socket cells, and local correlation of bristle polarity was weak (Figure 7D). Thus, the local domains of correlated asymmetric Pk^sple localization in ds knock-down socket cells depend on the presence of Pk^pk; Pk^sple alone is insufficient to facilitate local signaling between neighbors. Finally, removing Pk^sple in ds knock-down wings failed to significantly modify ds knock-down effects on the polarity of bristles (and also hairs) while removing both Pk^pk and Pk^sple does (Figure 7E,F, Figure 7—figure supplement 1A–D), confirming that ds knock-down affects the Pk^pk-mediated, rather than the Pk^sple-mediated, PCP signal for wing bristle (and hair) polarity. Taken together, these observations suggest that the locally correlated domains of polarity observed in ds knock-down wings depend on Pk^pk activity.

These and previous results indicate that Pk^pk is the principal isoform functioning in core PCP signaling during bristle polarization. They are most consistent with, though do not definitively show, that in wildtype, the Ds global signal directs orientation of core signaling such that Vang and Pk^pk localize to the proximal side (and incidentally colocalizing Pk^sple to the proximal side) to establish normal polarity. The alternative possibility is that Ds activity is permissive, and some other signal directs this orientation of Pk^pk-dependent core polarization. The proposal that Ds is instructive for orienting Pk^pk-dependent core signaling, while consistent with our prior interpretation of coupling between the Ft/Ds/Fj signal and Pk^pk-dependent core PCP signaling in wing hair polarization (Ma et al., 2003; Olofsson et al., 2014; Sharp and Axelrod, 2016; Yang et al., 2002), is at odds with other reports asserting that while under Pk^pk control, core PCP directionality is uncoupled from the Ft/Ds/Fj signal (Merkel et al., 2014; Ambegaonkar and Irvine, 2015). Rigorous testing of this hypothesis requires reorienting the Ft/Ds/Fj signal and assessing the isoform dependence of the response.

It was previously shown that reversing the gradient of Ds expression near the distal part of the wing under control of distal-less-GAL4 (dll >2 x-ds) reverses wing hair polarity (Harumoto et al., 2010). Assuming that hair polarity is determined by Pk^pk, for which ample evidence exists, and that it depends on core signaling, this result would demonstrate coupling of Pk^pk-dependent core PCP signaling to the Ds signal. We rigorously tested this assumption by testing the core signaling and Pk isoform dependence of this response (Figure 7G–K). dll >2 x-ds reverses polarity of a substantial swath of wing hairs, precisely in the region where the Dll expression gradient is expected to be steepest (Figure 7—figure supplement 1E). dll >2 x-ds, however does not reverse AWM bristle polarity, as it does not produce a proximal-to-distal expression gradient at the AWM. Because our results show that AWM bristle and wing hair polarization show indistinguishable responses to Ft/Ds and to core PCP manipulations, we propose that wing hairs are a suitable readout for this assay. We first asked whether dll >2 x-ds-driven hair polarity reversal depends on core module activity by removing Fz, and found that ectopic Ds-dependent reversal is blocked in a fz mutant background (Figure 7G,H). Furthermore, ectopic Ds re-orients the core PCP protein orientation (Figure 7—figure supplement 1F–I). These results rule out the possibility that ectopic Ds reverses polarity through a pathway that does not include the core PCP module. We then tested the Pk isoform dependence of reversal, and found that it is almost entirely abolished upon removal of Pk^pk (pk^pk or pk^pk-sple), but is largely unchanged upon removal of Pk^sple (pk^sple) (Figure 7G,I–K). This result decisively demonstrates that Pk^pk-dependent core PCP signaling in wing hair polarization is oriented by the Ft/Ds/Fj signal, and strongly suggests that the same coupling occurs during Pk^pk-dependent AWM bristle polarization.

Producing structures of defined chirality requires directional information on three Cartesian axes. Our results indicate that in determining bristle chirality, PCP provides directional information along the proximal-distal axis. The apical-basal axis is defined by the epithelium, while the dorsal-ventral axis is likely defined by the dorsal-ventral compartment boundary.

We have shown that the polarity of wing margin bristles (proximal or distal tilt) is determined by controlling the handedness of helical growth. The entwined shaft and socket cells undergo an apical clockwise or counterclockwise rotation that results in a left-handed or right-handed helical structure, placing the shaft to the proximal (wildtype) or distal (pk^pk mutant) side of the socket cell. The direction of rotation depends on PCP signaling among and between margin and socket cells. Helical cellular structures of defined handedness, such as the bristles resulting from properly directed rotation, have been noted in bacterial and plant species, but few examples have been described in animals.

The entwined twisting of the shaft and socket is a coordinated morphogenetic event, and the apparent stereotyped junctional rearrangement of additional margin cells suggests that at least some other cells are involved as well. We do not know in which cell or cells mechanics are regulated to drive this morphogenesis. One possibility is that an internal cytoskeletal mechanism induces the helical growth of the socket and/or shaft cells. Another possibility is that the side of the socket cell crescent marked by Pk at 24 hr is anchored, while the other side grows to wrap around the shaft, inducing junctional rearrangements and propelling the rotation of the apical portion of the shaft relative to the socket cell. Apical rotation could then cause twisting of the more basal portions of the socket cell, and could in turn direct the shaft to the corresponding side of the socket cell.

The precise location at which the PCP signal is required to determine rotation direction is unclear. Because we observe asymmetric core complexes at margin-socket cell junctions, but very little at shaft-margin or shaft-socket junctions, we hypothesize that interaction between the socket and surrounding margin cells is the essential determinant of rotation. PCP proteins at these junctions could control junctional dynamics, as is known to occur in other systems (Huebner and Wallingford, 2018). This will require further investigation.

Core PCP signaling participates in regulating rotation, as the magnitude of rotation is substantially impaired in the core mutants fz, dsh, vang and fmi. Nonetheless, we note that a small amount of clockwise rotation still occurs in these mutants. We hypothesize that tissue scale mechanical forces may drive this rotation, though we do not rule out the possibility that some other signaling activity may also be involved. Compared to other core proteins, the impact of removing Pk^pk on the magnitude of rotation is subtle. The clockwise rotation in pk^pk-sple mutants is only slightly less than in wildtype. This result is reminiscent of Pk^pk function in polarizing wing hairs: polarity in pk^pk mutants is strongly perturbed due to the presence of Pk^sple, while hair polarity in pk^pk-sple mutants is only weakly perturbed (Gubb et al., 1999). These findings are consistent with previous proposals that the core PCP mechanism retains a residual capacity to propagate some asymmetry in the absence of Pk (Strutt and Strutt, 2007; Lawrence et al., 2004; Adler et al., 2000).

The core PCP signal, in addition to executing directed rotation in response to Pk^pk or Pk^sple, coordinates polarity between neighboring bristles. Local correlation between rotation angles is strong when core signaling is intact, even in the absence of the Ft/Ds signal, but is weak when core signaling is disrupted. This is analogous to the proposed mechanism for locally coordinating polarity between adjacent wing hairs. It is important to note that the local polarity signal must pass through intervening margin cells to signal from bristle to bristle.

Our data suggest that whether the Pk^pk or Pk^sple isoform dominates to control the direction of PCP signaling depends not only on the relative amounts of each isoform, but also on the dynamics of expression and its effect on competition for participation in the core complex. During rotation of AWM bristle shaft-socket pairs, both Pk^pk and Pk^sple isoforms are detected at the apical junction of the socket with an inverse temporal relationship; high expression of Pk^pk decreases during rotation, while the initially undetectable level of Pk^sple protein increases. In these conditions, the system is controlled by Pk^pk, and both Pk^pk and Pk^sple localize proximally, thus orienting the core complex in its wildtype configuration. We hypothesize that Pk^sple is recruited by Pk^pk through their known ability to interact heterotypically (Ayukawa et al., 2014; Ambegaonkar and Irvine, 2015). This ability of Pk^pk and Pk^sple to colocalize has not been previously observed in wildtype conditions. Notably, however, in the wing, ectopic Pk^sple localization follows the expected position of Pk^pk when Ds and Dachs cues are removed, though each were not independently visualized (Ambegaonkar and Irvine, 2015). Conversely, Pk^sple overexpression was seen to recruit Pk^pk to the distal side of wing cells (Ayukawa et al., 2014), reversing hair polarity as it does bristle polarity. We suggest that the temporal expression pattern in the AWM allows the system to initiate polarization under Pk^pk control, and that the gradually accumulating Pk^sple colocalizes with Pk^pk rather than outcompeting established proximal localization. Because bristles in pk^sple mutant wings fully polarize, the proximal Pk^sple is inconsequential for normal bristle polarization.

In contrast, overexpression of Pk^sple, producing early and sustained high level expression, enables it to outcompete endogenous Pk^pk and reverse polarity by driving localization to the distal side through its interaction with Ds and Dachs, likely recruiting Pk^pk along with it. Similarly, in pk^pk mutants, endogenous Pk^sple, free from recruitment to the proximal side, localizes distally and reverses polarity. We infer that during the critical period for determining bristle rotation direction in wildtype, Pk^sple does not reach a sufficient level to outcompete Pk^pk and reverse the rotation.

That pk^pk mutation only reverses polarity of a region of distal bristles, whereas Pk^sple overexpression can reverse polarity of most or all AWM bristles, indicates that endogenous Pk^sple is only poised to act in a limited region of the margin. This may reflect subtle differences in the timing of its expression increase across the margin. Alternatively, it may reflect differences in the strength of the Ft/Ds/Fj signal across the margin. Our analyses do not have sufficient resolution to distinguish these possibilities.

Dynamic isoform expression appears to have important consequences for other aspects of wing development. Hair polarity is determined by Pk^pk (at around 32 hr apf), but it can be inferred that some Pk^sple is already present, as is evident from the difference between the hair polarity patterns of pk^pk and pk^pk-sple mutants (Gubb et al., 1999), and as confirmed by our expression analyses. We suggest that hair polarity does not fully reverse in pk^pk mutants either because levels of Pk^sple are not yet high enough or because expression is primarily in veins and at the AWM at the time hair polarity is fixed. In contrast, the polarity of ridges, established later in wing development [(Merkel et al., 2014); (Doyle et al., 2008) notwithstanding], depends on Pk^sple. We propose that by the time ridge polarity is determined, the amount of Pk^sple has increased and the amount of Pk^pk has decreased sufficiently to allow Pk^sple to exert control of ridge polarization. Though likely unimportant for normal development, the somewhat earlier expression of Pk^sple in veins relative to the intervein regions may contribute to polarity discontinuities observed in pk^pk mutant wings, especially around L3 (Gubb and García-Bellido, 1982; Hogan et al., 2011; Merkel et al., 2014). Ft-Ds polarity appears to also be distorted around veins (Merkel et al., 2014). Pk^pk and Pk^sple expression dynamics are likely at play in determining the PCP response in other tissues as well.

The idea that Ds controls the direction of core PCP signaling was first proposed by Adler based on wing hair polarity phenotypes (Adler et al., 1998). We subsequently studied the Ft/Ds/Fj system and similarly concluded that it directs core PCP protein localization in the wing (Ma et al., 2003), a Pk^pk-dependent process, and polarization of ommatidia in the eye (Yang et al., 2002), a Pk^sple-dependent process. We proposed that coupling in wing hair polarization is necessarily weak (Ma et al., 2003), and the more recently proposed model in which Ft/Ds/Fj orient microtubules to orient directional trafficking of Fz, Dsh and Fmi-containing vesicles (Matis et al., 2014; Olofsson and Axelrod, 2014; Shimada et al., 2006; Harumoto et al., 2010) is consistent with a weak coupling mechanism in Pk^pk-dependent processes. The finding of direct binding of Pk^sple to Ds and Dachs (Ayukawa et al., 2014; Ambegaonkar and Irvine, 2015) suggests a model for more direct and potentially stronger coupling of Pk^sple-dependent processes to the Ft/Ds/Fj system. The idea of coupling in Pk-dependent signaling has been controversial, and based largely on correlation, subsequent studies have led to the argument that Pk^sple-dependent core signaling is coupled, but Pk^pk-dependent signaling is uncoupled from the the Ft/Ds/Fj system (Merkel et al., 2014; Ambegaonkar and Irvine, 2015). Yet others have suggested that the Ft/Ds/Fj and core PCP systems always function in parallel rather than being coupled (Lawrence et al., 2007; Casal et al., 2006). Here, we report strong evidence that Pk^pk-dependent core PCP signaling is responsive to the Ds signal, at least in polarizing wing hairs. We propose that the same is the case in polarizing bristles that are controlled by essentially similar responses to Pk isoforms and to the Ft/Ds/Fj system.

In bristles, we directly observe the requirement for Ds to distally localize Pk^sple when Pk^pk is absent, confirming Pk^sple coupling. The evidence that Pk^pk-dependent core signaling is coupled to the upstream Ft/Ds/Fj signal is less apparent. In wildtype, correct bristle polarization requires Pk^pk to prevent reversal by recruiting Pk^sple to the proximal side, though as noted above, proximal Pk^sple plays no essential role. But absent the need to antagonize Pk^sple coupling, is there evidence that core signaling in the presence of just Pk^pk (pk^sple mutant) is coupled to Ft/Ds/Fj? When the Ft/Ds/Fj system is intact, Pk^pk localizes proximally, but without Ds or Ft, Pk^pk localizes proximally and distally in random domains, driving domains of correct and reversed rotation analogous to the random but locally correlated domains of hair polarity in ft or ds mutant wing tissue (Adler et al., 1998; Ma et al., 2003). The same random domains of bristle polarity are seen when only Pk^pk is available (ds knockdown in a pk^sple mutant). This result demonstrates that the Ft/Ds/Fj system is required for correct polarization of the core PCP system while solely under Pk^pk control, though it cannot distinguish a permissive from an instructive role. An instructive role is, however, concordant with its instructive role in directing Pk^pk-dependent wing hair polarity.

The proposal that Pk^pk-dependent core signaling is coupled to and responds to the Ft/Ds/Fj signal in bristle polarization might at first appear to conflict with the observation that properly oriented rotation proceeds to a significant extent in the absence of both Pk^pk and Pk^sple. This is explained by pointing out that our model for coupling invokes Ft/Ds/Fj directed microtubule-based transport of Fz and Dsh, but that the involvement of Pk^pk is indirect (Matis et al., 2014; Olofsson and Axelrod, 2014; Shimada et al., 2006; Harumoto et al., 2010). As have others, we propose that Pk^pk functions to amplify the asymmetry introduced by this transport, but that some asymmetry, and communication of polarity information between cells, can still occur in its absence (Strutt and Strutt, 2007; Lawrence et al., 2004; Adler et al., 2000). In other words, Ft/Ds/Fj coupling to Pk^pk-dependent core PCP signaling is not directed by Pk^pk, but rather, is permitted to occur because the Pk^sple-dependent mechanism is not operating to override it. Because core module function is required, this activity does not result from Pk isoform action influencing Ft/Ds/Fj output independent of core signaling, as has been recently suggested in another context (Casal et al., 2018). It is important to caution that the model for the relationship between Ft/Ds/Fj and core signaling presented here does not necessarily extend to their relationship in other tissues where their interactions may well be different, and that experiments done in other tissues may not be directly relevant to wing hair and AWM bristles.

The results presented here indicate that mapping the spatiotemporal dynamics of Pk isoform expression is essential to understanding how various developmental events can be differentially coupled to upstream global directional signals in a given tissue.

Chirality, or left-right laterality, is a key feature of many organs in invertebrates and vertebrates. In Drosophila, rotation of the gut and of the male genitalia occurs in a defined direction to produce such laterality. In vertebrates, rotation of the gut and heart tube also leads to left-right asymmetry in these organs. In many cases, PCP has been implicated in control of this lateralization (Blum and Ott, 2018). In the Drosophila hindgut, both core PCP and the Ft/Ds system play essential roles in directing normal dextral rotation (González-Morales et al., 2015). Though the forces that drive these organ rotations are not well understood, left-right asymmetries in actomyosin distribution, cell shape, and localization of other cellular structures, together with PCP dependence (Harris, 2018; Blum and Ott, 2018), indicate that chirality at the cellular level is an important determinant of rotational direction. Indeed, chirality of isolated cells from looping chick heart has been directly demonstrated (Ray et al., 2018). We therefore propose that regulation of chiral shaft-socket cell pair rotation may share much in common with the mechanisms that determine larger organ laterality, and its investigation could therefore yield insights that will enlighten understanding of organ rotation and laterality.

Drosophila melanogaster flies were grown on standard cornmeal/agar/molasses media at 25°C. FLP-on (using the actP >CD2>GAL4 construct for trans-gene expression) and FLP/FRT mitotic clones were generated by incubating third-instar larvae at 37°C for 1 hr. 36 to 48 hr later, white prepupae were collected and aged to the desired developmental time point prior to dissection and fixation.

Drosophila mutant alleles and transgenic stocks are described in the Key resources table and detailed chromosomes and genotypes are provided below. pk^pk-sple13 (FBst0044230), pk^pk-sple14 (Gubb, 1993), pk^pk30 (FBst0044229), pk^sple1 (FBst0000422), vang^A3 (Taylor et al., 1998), vang^stbm6 (FBst0006918), fz^R52 (Krasnow and Adler, 1994), dsh¹ (FBst0005298), UAS-pk^sple (FBst0041780), UAS-pk^RNAi (VDRC ID: 101480), UAS-fmi^RNAi (FBst0026022), UAS-fz^RNAi (FBst0034321), UAS-vang^RNAi (FBst0034354), UAS-ds^RNAi (FBst0032964), UAS-ds (Matakatsu and Blair, 2004), dll-Gal4 (FBst0030558), MS1096-Gal4 (FBst0008860), armP-fz::EGFP (Strutt, 2001), actP-vang::EYFP (Strutt, 2002), actP >CD2>vang::EYFP (Strutt, 2002), ci-Gal4 (Croker et al., 2006), UAS-mCherry (FBst0038424), actP >CD2>Gal4 UAS-RFP (FBst0030558).

Pupal wings were dissected at indicated developmental time points after puparium formation (apf). Pupae were removed from their pupal cases and fixed for 60–90 min in 4% paraformaldehyde in PBS at 4°C. Wings were then dissected and extracted from the cuticle, and were washed two times in PBST (PBS with 0.1% Triton X-100). After blocking for 1 hr in 5% Bovine serum Albumin in PBST at 4°C, wings were incubated with primary antibodies overnight at 4°C in the blocking solution. Incubations with secondary antibodies were done for 90 min at room temperature in PBST. Washes in PBST were performed three times after primary and secondary antibody incubation, and incubations in phalloidin (1:200 dilution) in PBST were done for 15 min followed by wash at room temperature before mounting if required. Stained wings were mounted in 15 μl Vectashield mounting medium (Vector Laboratories). Primary antibodies were as follows: goat polyclonal anti-Su(H) (1:200 dilution, Santa Cruz, sc-15183), mouse monoclonal anti-V5 (1:200 dilution, Thermo-fisher, R960-25), guinea pig polyclonal anti-Pk[C] (1:800 dilution, Olofsson et al., 2014), rat monoclonal anti-dEcad (1:200 dilution, DSHB). Secondary antibodies from Thermo Fisher Scientific were as follows: 488-donkey anti-mouse, 488-goat anti-guinea pig, 546-donkey anti-goat, 633-goat anti-guinea pig, 633-goat anti-rat, 647-donkey anti-mouse. Alexa 635 and Alexa 350 conjugated phalloidin were from Thermo Fisher Scientific.

Adult wings were dissected and washed with 70% EtOH and mounted in DPX (Sigma) solution. All adult wings were imaged on a Nikon Eclipse E1000M equipped with a Spot Flex camera (Model 15.2 64 MP). All immunofluorescence images were taken with a Leica TCS SP8 AOBS confocal microscope and processed with LAS X (Leica) and Adobe Photoshop. For three dimensional wing margin images, 50 to 100 z-stacks, each with 0.2 μm thickness, were collected and combined using 3D reconstitution software (Leica). Scale bars are not provided for three dimensional images due to errors introduced by perspective, but approximate scale can be inferred from related two- dimensional images. To measure the apical rotation angles of socket cells, a horizontal line linking centers of circles around apical surfaces of socket cells was drawn, and perpendicular lines intersecting the center of each socket cell apex were drawn (black lines in Figure 3C). Vectors from each socket cell center passing through the center of the apical opening of the socket circles (blue vectors in Figure 3C) were drawn and angles between the vertical lines and the vectors were measured with Image J software. Statistical analysis was performed and rose plots generated using Oriana four software. Comparisons were made using Student’s t-test and p values are reported. Summary statistics are provided in Table 2. For qualitative results such as expression patterns, a minimum of 20 biological replicates from at least two independent experiments were examined and representative images are shown.

To generate the donor plasmid with homology arms of the pk^pk genomic sequence and the V5::3Xmyc::APEX2 tag sequence, a 1.5 kb 3’ homology arm (HR2) flanking the pk^pk gRNA2 cleavage site was amplified and assembled into the pDsRed-attp (Addgene, 51019) plasmid cut with SapI to make the pDsREd-attP-pk^pkHR2 plasmid.

To generate the donor plasmid for tagging pk^pk, three DNA fragments including 5’ 1.5 kb homology arm (HR1; containing a 1.2 kb homology arm flanking the pk^pk gRNA1 cleavage site and a 5’ 0.3 kb sequence of the start codon), V5::3Xmyc::APEX2 tag with a linker sequence, and the fragment starting from the start codon of pk^pk to the cleavage site targeted by the pk^pk gRNA2, were assembled into the pDsREd-attP-pk^pkHR2. To prevent the donor sequence from being cleaved by Cas9, a point mutation was introduced in the PAM sequence of the HR1 using the NEB point-mutagenesis kit after sub-cloning the fragment into the pCR-Blunt-II-TOPO vector (Thermo-Fisher, K280002). The HR1 fragment bearing the mutant PAM sequence was then amplified for the assembly process. All three fragments were assembled into the pDsREd-attP-pk^pkHR2 plasmid cut with EcoRI and NheI.

To generate the donor plasmid for tagging pk^sple, similar strategies were applied. Briefly, 1.2 kb 5’ homology arm containing the mutant PAM sequence, the V5::3Xmyc::APEX2 tag with a linker sequence, and the fragment from the start codon of pk^sple to the cleavage site of pk^sple gRNA2 were assembled into the pDsREd-attP-pk^spleHR2 (bearing the 1.25 kb 3’ homology arm, HR2) plasmid.

The donor plasmids containing the tag sequence and pk^pk homology, or pk^sple homology, arms, were sequenced and then injected into the stable transgenic flies expressing two pk^pk gRNAs, or pk^sple gRNAs, and nosCas9, respectively, to generate recombinants. DsRed signal in the fly eyes was monitored for selecting the recombinants by BestGene, and dsRed and flanking sequences were removed by the Cre-Lox site-specific recombination method. The resulting modified alleles are referred to in the text as V5::Pk and V5::Sple for simplicity.

Third-instar larval wing discs and pupal wings at appropriate developmental stages were dissected and lysed in protein loading buffer. Lysates from eight discs or wings were loaded per lane for SDS-PAGE analysis and western blots were performed using standard procedures. Antibodies: Guinea pig polyclonal anti-Pk[C] (1:1000 dilution, the same antibody used for immunostaining), mouse monoclonal anti-V5 (1:2000 dilution, the same antibody used for immunostaining), mouse monoclonal anti-γ-Tubulin (1:1000 dilution, Sigma-Aldrich, T6557). Secondary antibodies were Peroxidase-conjugated goat anti-guinea pig (1:10000) and goat anti-mouse (1:10000) antibodies (both from Jackson Immuno Research), and detection used SuperSignal West Pico Chemiluminescent Substrate (Thermo-Fisher, 34080)

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank Helen McNeill, Lucy O’Brien and members of the Axelrod lab for constructive comments.

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocol (#11840) of Stanford University.

© 2020, Cho et al.

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