Wound size does not affect the mechanism of repair used

We observed both dynamic cellular protrusions and formation of an actomyosin cable during the coalescence phase, suggesting that Drosophila embryos may be able to repair lesions using more than one mechanism. One of the factors proposed to affect the mechanism by which wounds will be repaired is wound size. To determine if this was a deciding factor in the Drosophila embryo, we examined repair in different sized wounds (Fig. 2A–I, Table 1). Wounds fell in three different size ranges (area determined at fully expanded wound): (i) small: wound area <500 µm²; (ii) medium: wound area ranging from 500-1200 µm²; and (iii) large: wound area >1200 µm². Much larger sized wounds (>2500 µm²) can undergo successful repair, but were not used here since they often trigger an accompanying stress response complicating subsequent analyses. We found that in wild-type embryos, wound repair kinetics followed the same trends (see percentage of time in each phase) and different sized wounds displayed similar phenotypic repair dynamics (i.e. using an actin cable and actin-rich protrusions) (Fig. 2A–I; Table 1; and data not shown).

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Fig. 2.

Parameters of epithelial wound repair. (A–I) Parameters of the epithelial wound response (all results are given as means ± s.e.m.). (A) Effects of wound size on wound repair dynamics (small, n=5; medium, n=10; large, n=5). Analysis of each phase of repair: expansion and coalescence by area (B,D,F) and duration (C,E,G); contraction phase by area (H) and duration (I). (J–L) Effects of embryo mounting techniques on wound repair. Single slice micrograph at the plane of the wound in membrane (J) and coverslip (K) mounted embryos expressing actin (sGMCA). (L) Analysis of wound repair dynamics using membrane (blue) and coverslip (red) mounting. Scale bar: 20 µm.

We were surprised, however, to find that the method of mounting the embryos for wounding and imaging greatly affected the outcome of the wound repair dynamics (Fig. 2J–L). To perform laser wound assays, embryos were initially mounted either: (i) in series 700 halocarbon oil between a hydrophilic gas permeable membrane and a coverslip (a popularly used mounting method; Wood and Jacinto, 2005), or (ii) on a glue-painted coverslip then covered with halocarbon oil (Foe et al., 2000). The epithelial wound response was highly reproducible using glue mounting (Fig. 2L). However, embryos mounted using the membrane exhibited a highly variable repair response and in some cases even failed to heal (Fig. 2L). This interference with wound repair is likely caused by the slight compression introduced when embryos are sandwiched between the membrane and coverslip (Fig. 2J,K). Thus, our wound assays were typically conducted using medium size wounds with embryos mounted on glue painted coverslips. Under these conditions, wound repair was completed on average by 50 minutes (Table 1).

Myosin is required for the contraction of the actomyosin purse string

Actomyosin-powered purse string contraction is a mechanism common to multiple morphogenetic movements in a variety of organisms (Young et al., 1993; Williams-Masson et al., 1997; Vavylonis et al., 2008), and has been suggested to power wound closure in Drosophila embryonic epithelium wounds (Wood et al., 2002). To further understand the role of the actomyosin cable, we examined the function of myosin II in vivo and its contribution to the wound repair process. We first monitored the dynamics of myosin II recruitment relative to actin by examining embryos co-expressing a myosin regulatory light chain (sqh) fusion to GFP (sqh-GFP) and actin (sChMCA). Myosin II can be detected at the wound edge 5 minutes after wounding, overlapping with actin accumulation along the leading edge (Fig. 3A–B′; supplementary material Movie 1B). During contraction, actin and myosin co-localized to form a robust actomyosin purse-string in both surface and orthogonal planes. A striking feature of this accumulation was the complete absence of myosin II from the actin-rich protrusions (Fig. 3A–B′), suggesting that myosin II is only associating with actin as part of the supracellular cable.

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Fig. 3.

Myosin II is required for assembly of an actin purse string. (A–B′) Time-lapse series of surface projections (A,A′) and cross-sections (A″) of a stage 15 embryo expressing myosin and actin markers (sqh-GFP and sChMCA). Myosin accumulates at the leading edge of epithelium wounds overlapping with the actin cable. (B,B′) High magnification views (white squares in A) showing myosin and actin overlapping at the leading edge. Myosin II is absent in the protrusions (arrows). (C,C′) Time-lapse series of stage 15 zip¹ mutant embryo expressing an actin marker (sGMCA), and showing incomplete actin cable assembly. Surface projections (C) and cross-sections (C′) are shown. (D,E) Quantification of wound area over time (wild type, n=10; zip¹, n=4; results are given as means ± s.e.m.) for all phases of wound repair (D) and an expansion of the first 20 minutes (E). (F) High magnification (100×) views of protrusions in wild-type and zip¹ wounds at time-points representing 100, 75 and 50% of the maximum wound area. (G) Quantification of the area of protrusions (wild type, n=4; zip¹, n=4), P-values are indicated. Scale bars: 20 µm (A,A′,C) and 10 µm (A″,C′,F).

Given myosin dynamics in response to wounding, we anticipated that mutations affecting myosin II would impair actomyosin purse string assembly and contractility without affecting the actin-rich protrusions. In zipper¹ (zip¹) homozygous mutants, which disrupt the myosin II heavy chain gene, an actomyosin purse string is not assembled at the leading edge of the wound (Fig. 3C,C′; Table 2; supplementary material Movie 2B). The wound edges were irregular, with actin accumulated in patches that extended over a few cells and particularly at cell-cell junctions, but never forming the thick continuous cable that encircles wounds in wild-type embryos (Fig. 3C,C′). Interestingly, despite the lack of a contiguous actin cable, wounds in zip¹ mutant embryos healed, albeit with a severe delay especially in the contraction phase (P=0.0051; Fig. 3D,E; Table 2). In vivo analysis showed that wound closure was achieved by the actin-rich cellular protrusions (Fig. 3C,C′,F; supplementary material Movie 2B). Quantification of the wound area covered by protrusions showed that zip¹ embryos displayed three times more protrusions than wild-type embryos at similar stages of repair (Fig. 3G). This increase could be detected from the coalescence phase (100% of max wound area) (Fig. 3F,G). These protrusions pulled the wound closed by capturing protrusions from adjacent or opposing cells and tugging these areas closer together. As a consequence, reduction of the wound area occurred in jumps (Fig. 3D; supplementary material Fig. S1C). Our data suggests that the contractile force of the actomyosin purse string was necessary for the rapid closure of the wound during the contraction phase, but in the absence of this component, both local and long-distance zippering by dynamic filopodia and lamellipodia can mediate wound closure.

Cadherin-based adherens junctions are necessary to mediate the actomyosin purse string contraction

In order for the actomyosin purse string to contract to close a wound it needs to be linked from cell to cell to generate a supracellular cable. Cadherin-based adherens junctions are a major site of cell-cell adhesion and actomyosin network anchoring to the plasma membrane within a cell (reviewed in Tepass et al., 2001; Hartsock and Nelson, 2008). Analysis of epithelium wound repair in embryos expressing markers for E-Cadherin (DE-Cadherin-GFP) and actin (sGMCA) showed that E-Cadherin accumulated at the apical most side of the leading edge cells and in particular at the sites of lateral cell-cell junctions, consistent with its reported role (Fig. 4A,A″′; supplementary material Movie 1C) (Wood et al., 2002). By midway through the contraction phase, cadherin expression was largely lost along the apical most side of leading edge cells, remaining at the sites of lateral cell-cell junctions (Fig. 4B,B′). Interestingly, we found that immediately following injury, some of the cells at the wound site retained DE-Cadherin expression but lacked actin (Fig. 4A). These cells slowly disappeared from the surface of the embryo as the leading edge cells moved forward to close the wound (Fig. 4A,A′).

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Fig. 4.

Intact adherens junctions are required to anchor the actomyosin cable. (A–B′) Time-lapse series of surface projections (A–A″,B,B′) and cross-sections (A′″) in wild-type embryos expressing DE-cadherin-GFP and actin (sChMCA). Cells retaining E-cadherin but lacking actin are indicated (white arrowheads). (B,B′) High magnification views (white squares in A) showing actin and E-cadherin overlaps at the leading edge junctions. (C–D) Wound repair in a shg^k03401 mutant embryo expressing an actin marker (sGMCA). Time-lapse series of surface projections (C,D) and cross-sections (C′). High magnification views of shg^k03401 embryos showing repair by localized protrusion zippering between neighbor leading edge cells (D, white brackets). (E,F) Quantification of wound area over time (wild type, n=10; shg^k03401, n=6; results are given as means ± s.e.m.) for all phases of wound repair (E) and an expansion of the first 20 minutes (F). (G–H) Time-lapse series of surface projections (G,H) and cross-sections (G′) of an ed^M/Z mutant embryo expressing an actin marker (sGMCA). ed^M/Z protrusions contact one another, resulting in local contractions of the wound edge (H, arrows). (I,J) Quantification of wound area over time (wild type, n=10; ed^M/Z, n=5) for all phases of wound repair (I) and an expansion of the first 20 minutes (J). (K–L′) ed^M/Z mutants assemble partial actomyosin cables at the wound leading edge. Myosin II (α-non muscle myosin, MHC) accumulates at the wound edge of wild-type (K,K′) and ed^M/Z mutants (L,L′) overlapping with actin (phalloidin). Scale bars: 20 µm (A–A″,C,G); 10 µm (A″′,C′,D,H,G′); and 5 µm (K–L′).

Given the accumulation of DE-Cadherin at the sites of cell-cell junctions and its overlap with actin accumulation, perturbations in the levels of cadherin would be expected to disrupt cable function and possibly its assembly. Cadherin loss of function mutants (shotgun; shg^k03401) showed defects in wound repair during the contraction phase: actin cable assembly was impaired leading to initially jagged wound boundaries and irregularly shaped wounds (Fig. 4C,C′,E,F; supplementary material Fig. S1A; Table 2). Remarkably, wound closure was eventually achieved by non-uniform contraction of leading edge areas (a few cells wide) that accumulated actin. These areas of localized pinching often corresponded with cells exhibiting active protrusions (Fig. 4D; supplementary material Movie 2C). Our data shows that DE-Cadherin is required for both the assembly and stability of the actin purse-string at the wound leading edge.

Echinoid is required for actomyosin purse string assembly and stabilization

Another adherens junction-associated cell adhesion molecule, the Drosophila nectin ortholog Echinoid (Ed), has been shown to interact with Canoe (afadin) and Jaguar (unconventional myosin VI) to modulate the accumulation of actin regulators at the leading edge during dorsal closure (Wei et al., 2005; Laplante and Nilson, 2006; Lin et al., 2007; Chang et al., 2011; Laplante and Nilson, 2011). In this context, Ed has been proposed to mediate cell sorting by promoting actomyosin cable formation and filopodial protrusions in the dorsal most leading edge cells. In the context of epithelial wound repair, we anticipated that loss of Ed might prevent epithelial wound repair by destabilizing adherens junction complexes, disrupting actomyosin purse string assembly and stabilization, and by reducing cellular protrusions from leading edge cells. Consistent with this, ed^M/Z mutant embryos failed to form a continuous actomyosin purse-string at the leading edge of wounds (Fig. 4G,G′,K–L′), and exhibited significant delays in both the coalescence and contraction phases (P<0.0001 and P=0.0005, respectively) (Fig. 4I,J; Table 2; supplementary material Fig. S1A and Movie 2D). However, similar to cadherin mutants, wound closure took place by local contraction of actin patches and protrusions. Indeed, we observed protrusions that joined with those from nearby and/or opposing cells to pinch the wound shut (Fig. 4H). Thus, Ed in the context of wound repair may be required for the stabilization of adherens junctions needed to link the actomyosin cable intercellularly at the wound leading edge cells.

Actin-rich protrusions are present throughout the epithelial repair process

Previous studies suggested that an actomyosin cable was needed to close the edges of the wound until the fronts were close enough for cellular protrusions to reach across and knit closed the small remaining hole (Wood et al., 2002). In contrast to previous reports, we observed filopodia and lamellipodia throughout the wound repair process in wild-type embryos making contact with other protrusions (Fig. 1I–J′; Fig. 4B′). To determine the role of cellular protrusions during the contraction phase when the actomyosin cable is thought to be the major mechanism for closing the hole, we followed the dynamics of cellular protrusions during the wound repair process. In particular, we examined two membrane markers: GFP-PH-PLC, the pleckstrin-homology domain of PLC fused to GFP, which specifically binds to the phosphoinositidine PI(4,5)P2 and is enriched in the apical side of epithelial cells (Pinal et al., 2006), and Venus-GAP43, the palmitoylation signal of the growth-associated protein 43 fused to Venus fluorescent protein, which tightly anchors to the plasma membrane (Mavrakis et al., 2009). In addition, we examined a marker for microtubules as they have previously been reported to be reorganized in response to injury (Etienne-Manneville, 2004).

Cells at the leading edge of the wounds show highly dynamic filopodia and lamellipodia enriched in both membrane markers, particularly when the PH-PLC reporter was used (Fig. 5A,B). These cellular extensions reach across the wound to make both nearby and long-distance connections to the protrusions of other cells (Fig. 5A,B; supplementary material Movie 3). In addition, GAP43 was observed to accumulate in the cell-cell contacts (Fig. 5C–C″).

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Fig. 5.

Dynamic protrusions are assembled at the leading edge of epithelial wounds. (A–C″) Time-lapse series of surface projections (A,A″,B,C,C″) and cross-sections (A′,C′) of embryos expressing the membrane reporters PH-PLC-GFP (A–B) or Venus-GAP43 (C–C″). High magnification view of protrusions in embryos expressing PH-PLC-GFP (A″,B) and Venus-GAP43 (C″). Membrane in protrusions (arrows) and accumulation at cell-cell junctions (arrowheads) are indicated. (D–F) Time-lapse series of embryos expressing GFP-α-tubulin in the epithelium, showing microtubules in cell protrusions (arrows). High magnification views in E,F. (G,G′) Time-lapse series of surface projections (G) and cross-sections (G′) of a stage 15 eb1² embryo expressing an actin marker (sGMCA) showing delayed actin cable assembly. (H,I) Quantification of the wound area over time (wild type, n=10; eb1², n=6; results are given as means ± s.e.m.) for all phases of wound repair (H) and an expansion of the first 20 minutes (I). Scale bars: 20 µm (A,C,D,G) and 10 µm (A′,A″,B,C′,C″,E,F,G′).

Time-lapse movie analysis of Drosophila embryos expressing GFP-α-tubulin in the epithelium shows that, unlike actin, microtubules do not accumulate at the leading edge of the wound (Fig. 5D–F). However, consistent with previous observations during the dorsal closure process (Jankovics and Brunner, 2006; Liu et al., 2008), microtubules are recruited to the filopodial protrusions assembled by the leading edge cells (Fig. 5D–F). We wounded embryos mutant for the microtubule capping protein EB1 (eb1²), which disrupts microtubule dynamics (Elliott et al., 2005). Interestingly, we observed defects only in the coalescence phase, where eb1² mutant embryos showed a significant delay before beginning contraction (P=0.045; Fig. 5G–I; Table 2). We are not able to rule out, however, that disruption of microtubule dynamics in this context is indirect and may not affect cellular protrusions per se, but rather affect repair during the coalescence phase by impairing the recruitment of the repair machineries/components thereby delaying initiation of the contraction phase.

Actin-rich protrusions are required for embryo epithelial wound closure

To further investigate the role of dynamic cellular protrusions, we examined the function of the Cdc42 small GTPase in vivo and its contribution to the wound repair process. Cdc42 constitutive active or dominant negative mutants have been shown to regulate filopodial assembly in tissue culture cells and in both dorsal closure and wound repair in Drosophila (Nobes and Hall, 1995; Jacinto et al., 2000; Wood et al., 2002). We first generated a ChFP-Cdc42 reporter to follow Cdc42 localization in vivo simultaneously with actin (ChFP-Cdc42; SGMCA) (Fig. 6A–A″). Throughout wound repair, Cdc42 did not accumulate at the wound leading edge or in the actin-rich protrusions, suggesting that upon wounding Cdc42 subcellular localization does not change significantly.

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Fig. 6.

Cdc42 is required for normal protrusion activity and, together with Myosin II, for proper epithelial repair. (A–A″) Confocal time series following wound repair in stage 15 embryos coexpressing ChFP-Cdc42 (A′) and actin (sGMCA). (B,B′) Time-lapse series of wound repair in Cdc42⁴/Cdc42⁶ mutant embryos expressing an actin marker (sGMCA). Notice the lack of protrusions at the leading edge cells. (C,D) Quantification of the wound area over time (wild type, n=10; Cdc42⁴/Cdc42⁶, n=5; results are given as means ± s.e.m.) for all phases of wound repair (C) and an expansion of the first 20 minutes (D). (E) High magnification views (100×) showing the protrusions at the leading edge cells in wild-type and Cdc42⁴/Cdc42⁶ embryos at 100, 75 and 50% maximum wound area. (F) Quantification of the area of protrusions, P-values are indicated (wild type, n=4; Cdc42⁴/Cdc42⁶, n=4). (G,G′) Time-lapse series of wound repair in zip¹ Cdc42^RNAi double mutant embryos followed with an actin marker (sGMCA). Actin cable assembly, as well as cellular protrusions, are severely disrupted. (H) Quantification of the wound area over time (wild type, n=10; zip¹, n=4; Cdc42⁴/Cdc42⁶, n=5, zip¹ Cdc42^RNAi, n=4; results are given as means ± s.e.m.). Scale bars: 20 µm (A,A′,B,G) and 10 µm (A″,B′,E,G′).

To modulate the levels of Cdc42, we used the heteroallelic combination Cdc42⁴/Cdc42⁶. These Cdc42 loss of function mutant embryos exhibit severe developmental defects as previously reported (Genova et al., 2000), where epithelial cells fail to elongate into columnar shaped cells, resulting in failure of germ band retraction and, in most cases, random holes appeared in the epithelium. Upon wounding, Cdc42⁴/Cdc42⁶ embryos were not significantly delayed in the expansion or coalescence phases (Fig. 6B–D; Table 2). By contraction, these wounds became quite rounded consistent with the presence of circumferential tension provided by a functional actomyosin cable (Fig. 6B,B′; supplementary material Fig. S1B and Movie 2E). Strikingly, we observed a severe reduction of filopodia and lamellipodia protrusions in the leading edge cells throughout the wound repair process: Cdc42⁴/Cdc42⁶ mutant embryos displayed three times less filopodia/lamellipodia than wild-type controls (Fig. 6E,F). As a consequence, these embryos spend 95 minutes in the contraction phase, over double the time spent by wild-type embryos (Fig. 6D; Table 2). As previously described, Cdc42⁴/Cdc42⁶ mutant embryos are unable to seal the wound during the final closure phase and a small hole persists (n=4/5 embryos) (Fig. 6B) (Wood et al., 2002). Our data shows that the contractile force of the actomyosin cable was sufficient to draw the wound closed, however the kinetics of this phase were not the same as those observed for repair in wild-type embryos, indicating that cellular protrusions are playing an active role in the contraction phase.

Wound repair occurs in distinct phases

A major observation while studying embryo epithelial wound healing in vivo is the existence of defined phases occurring at characteristic intervals along the wound repair process, and requiring specific molecular components. The expansion phase encompasses the initial clearance of the wound area and the establishment of the wound leading edge. Interestingly, none of the mutants we examined significantly affected this phase, suggesting that the factors affecting expansion may be inherent to the tissue. Notably, the expansion phase was not affected in zip mutant embryos, despite the epithelial cells in this mutant being smaller on their apical surfaces. In addition, in severe E-cadherin (shg²) mutant embryos in which only non-continuous patches of epithelium exist by later stages of development, wounds expand with normal dynamics, precluding morphogenetic tension as the explanation for the initial wound expansion (data not shown). We anticipate that mutants for components of the mechanosensory system and/or signals that initiate the wound repair response will impair the expansion phase.

We initially hypothesized that the coalescence phase corresponded to a passive transition where the forces exerted by the expanding wound were gradually overcome by the contraction machinery. However, the delay we observed in eb1 mutants is consistent with this phase being active. Furthermore, the coalescence phase is not extended in all mutants affecting the actomyosin cable, as would be expected if the contractile forces needed to overcome expansion are impaired.

The majority of wound closure is achieved during the contraction phase, as the wound is actively closed by a combination of cable contraction and cellular protrusions. The wound area exhibits exponential rather than linear reduction, consistent with a steady rate of wound margin ingression towards the center. Interestingly, in mutants lacking the actomyosin cable, the wound area is reduced in a more stepwise fashion corresponding to local zippering events. This suggests that the circumferential tension of the actomyosin cable not only provides direct force for repair, but also transmits the force generated by local zippering around the wound edge.

The final stage of repair is closure, in which filopodia and lamellipodia on opposing epithelial faces contact and engage to fuse the wound fronts and restore the continuity of the epithelium. Consistent with previous reports, our genetic analysis shows that the Cdc42 small GTPase is crucial in this phase of the process, by modulating the formation of polarized actin-rich protrusions (Wood et al., 2002). It will be interesting to see if trafficking of adhesion molecules, or perhaps integrins, along with filopodia are necessary for the protrusions to mediate epithelial fusion and the formation of new cell-cell junctions.

Dynamic cellular protrusions play a role throughout the wound repair process

In the absence of a functioning actomyosin cable, epithelium wounds heal by extension and zippering of actin-rich cellular protrusions. Previous studies have suggested that interactions between filopodia and lamellipodia of neighboring cells were only responsible for wound closure during the final steps of repair, when the edges of the wound were close to one another (Wood et al., 2002). In their study, wounding of Rho1 small GTPase embryos, in which the actomyosin cable is disrupted, showed a two-hour delay in repair during which no changes were observed in the leading edge cells, followed by the wound closing with normal wound kinetics mediated by local zippering (Wood et al., 2002). In contrast, our in vivo studies show that actin-rich protrusions are present throughout the wound repair process, from the onset of the coalescence through the closure phase. These apically-localized protrusions are highly dynamic, as observed with actin and plasma membrane markers, and reach across the wound fronts throughout the repair process to contact neighboring cells, as well as the overlying vitelline membrane. When we disrupted the actomyosin cable using myosin (zip) and E-cadherin (shg) mutants, we find that wound repair is significantly delayed, and showed that actin-rich protrusions are used for contraction in these mutants. Indeed, we observed long-range events where filopodia reaching far across the middle of the wound made contact and pulled the wound closed at the middle. Consistent with this, we observe a delay in contraction when the ability to form cellular protrusions, but not a functional actomyosin cable, was impaired using Cdc42 mutants (Table 2). The results observed with the Cdc42 mutants highlight two important aspects of the mechanisms involved in the repair process. First, protrusions are necessary for normal wound repair kinetics during contraction. Second, intact protrusions are crucial for the closure phase. In the absence of protrusions, the wound fronts fail to fuse indicating that the contractile force of the actomyosin cable cannot compensate for the lack of protrusions in the final steps of wound closure.

Generation of ChFP-Cdc42 and CyoChFP constructs and transgenics

ChFP-Cdc42 is a fusion of ChFP with the Cdc42 cDNA, expressed under the control of the spaghetti squash (sqh) promoter. The construct was generated as follows: the Cdc42 ORF was amplified by PCR from DGCr1 cDNA HL08128, then cloned 3′ of ChFP as a EcoRI and XbaI fragment. The ChFP-Cdc42 fusion was PCR amplified and cloned into the StuI and XbaI sites of the pSqh5′+3′UTR plasmid (Abreu-Blanco et al., 2011). To generate CyO-ChFP and TM3-ChFP balancers, ChFP was cloned into the StuI and XbaI sites of the pSqh5′+3′UTR plasmid (Abreu-Blanco et al., 2011). These constructs were used to make germline transformants as previously described (Spradling, 1986). The resulting transgenic lines were mapped to a single chromosome and shown to have non-lethal insertions.

RNA interference (RNAi) assays

To generate the Cdc42 double strand RNA (dsRNA), the template was amplified by PCR using primers that include the T7 promoter sequence. The sequence used for the Cdc42 dsRNA was selected based on previously reported RNAi lines by the TRiP stock center (DRSC25134). The dsRNA was synthesized and injected into embryos as previously described, at a final concentration of 2.5 µM (Magie et al., 2002; Magie and Parkhurst, 2005). Embryos were injected and aged at room temperature for 12–14 hours before conducting the wound healing assays. Control (sGMCA) and zip¹ (sGMCA, ChFP balancer) were injected with the Cdc42 dsRNA, generating Cdc42^RNAi and zip¹ Cdc42^RNAi embryos, respectively. Control (buffered injected) zip¹ was also included in the assay.

Immunofluorescence

Stage 15 laser wounded embryos were recovered, fixed with 37% paraformaldehyde/heptane for 5 minutes, and hand devitellinized. Immunofluorescence was performed as described previously (Abreu-Blanco et al., 2011) with anti-nonmuscle myosin antibody (1500; Young et al., 1993) and anti-rabbit Alexa Fluor 568 (11000; Invitrogen). Alexa-Fluor-488-labeled phalloidin (Invitrogen) was used at 5 Units/assay and added with the secondary antisera. Samples were mounted in SlowFade Gold (Invitrogen/Molecular Probes).

Confocal fluorescent microscopy

Confocal microscopy was performed using a Zeiss LSM-510M microscope (Carl Zeiss Inc., Jena, Germany) with excitation at 488 nm or 543 nm, and emission collection with BP-500-550 or LP560 filters, respectively. A Plan-Apochromat 20× 0.75 dry objective was used for imaging. Images were processed in ImageJ (http://rsb.info.nih.gov/ij/), and assembled with Canvas 8 software (Deneva Systems, Inc.).

Live imaging

All imaging was performed at room temperature (23°C). Stage 15 embryos were hand dechorionated, dried for 5 minutes then transferred individually with forceps onto strips of glue dried onto No. 1.5 coverslips, and covered with series 700 halocarbon oil (Halocarbon Products Corp.) (Foe et al., 2000). The following microscopes were used: (i) Nikon TE2000-E stand (Nikon Instruments, Melville, NY), with 40× 1.4 NA objective lens, controlled by Volocity software (v.5.3.0, PerkinElmer, Waltham, MA). Images were acquired with 491 nm and 561 nm lasers, with a Yokogawa CSU-10 confocal spinning disc head equipped with a 1.5× magnifying lens, and a Hamamatsu C9100-13 EMCCD camera (PerkinElmer, Waltham, MA). (ii) UltraVIEW VoX Confocal Imaging System (PerkinElmer, Waltham, MA), in a Nikon Eclipse Ti stand (Nikon Instruments, Melville, NY), with 60× 1.4 NA or 100× 1.4 NA objective lens and controlled by Volocity software (v.5.3.0, PerkinElmer, Waltham, MA). Images were acquired with 491 nm and 561 nm, with a Yokogawa CSU-X1 confocal spinning disc head equipped with a Hamamatsu C9100-13 EMCCD camera (PerkinElmer, Waltham, MA). (iii) Nikon LiveScan Swept Field Confocal (for Nikon by Prairie Technologies Inc., Middleton, WI) mounted on a Nikon Eclipse Ti (Nikon Instruments, Melville, NY); with 60× 1.4 NA objectives lens, using the NIS-Elements AR 3.0 as acquisition software (Nikon Instruments, Melville, NY). Images were acquired with a 491 nm laser, and a Photometrics QuantEM: 512SC EMCCD camera (Photometrics, Tucson, AZ). All images acquired with a 40×or 60×objective lens are 25 µm stacks/0.5 µm steps, for the 100×images the stacks correspond to 1.5 µm/0.25 µm steps.

Laser wounding

Laser ablation experiments used the Photonic Instruments Micropoint® Computer Controlled system (Photonic Instruments, St Charles, IL), as previously described (Abreu-Blanco et al., 2011).

We thank Parkhurst laboratory members for their interest, advice and comments on the manuscript. We are very grateful to D. Brunner, R. Fehon, R.E. Karess, D.P. Kiehart, J. Lippincott-Schwartz, L. Nilson, H. Oda, F. Pichaud, J.P. Vincent, and the Bloomington/Kyoto Stock Centers for flies and other reagents used in this study. We thank the M. J. Murdoch Charitable Trust for the spinning disk microscope used for imaging.