Patterned cortical tension mediated by N-cadherin controls cell geometric order in the Drosophila eye

Abstract
Introduction
Results
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Materials and methods
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Abstract

Adhesion molecules hold cells together but also couple cell membranes to a contractile actomyosin network, which limits the expansion of cell contacts. Despite their fundamental role in tissue morphogenesis and tissue homeostasis, how adhesion molecules control cell shapes and cell patterns in tissues remains unclear. Here we address this question in vivo using the Drosophila eye. We show that cone cell shapes depend little on adhesion bonds and mostly on contractile forces. However, N-cadherin has an indirect control on cell shape. At homotypic contacts, junctional N-cadherin bonds downregulate Myosin-II contractility. At heterotypic contacts with E-cadherin, unbound N-cadherin induces an asymmetric accumulation of Myosin-II, which leads to a highly contractile cell interface. Such differential regulation of contractility is essential for morphogenesis as loss of N-cadherin disrupts cell rearrangements. Our results establish a quantitative link between adhesion and contractility and reveal an unprecedented role of N-cadherin on cell shapes and cell arrangements.

https://doi.org/10.7554/eLife.22796.001

Introduction

Cells acquire different shapes and arrangements to form tissues, depending on their functions and microenvironment. During tissue morphogenesis, cells actively form and remodel their cell contacts, generating forces to drive various morphogenetic events (Lecuit and Lenne, 2007). In epithelia, cell division (Herszterg et al., 2013; Guillot and Lecuit, 2013; Founounou et al., 2013), cell intercalation (Bertet et al., 2004; Blankenship et al., 2006) and cell delamination (Marinari et al., 2012) are basic mechanisms of morphogenesis, which all involve gain or loss of cell contacts (Heisenberg and Bellaïche, 2013). Two systems contribute to changes in cell contacts: Cadherin complexes and actomyosin networks (Harris, 2012; Baum and Georgiou, 2011).

At the level of a single cell contact, formation of cadherin-cadherin bonds favors contact expansion. Actomyosin contractility acts antagonistically by reducing cell contact size (Lecuit and Lenne, 2007; Winklbauer, 2015). There is numerous evidence in vivo that shows actomyosin-generated tension regulates cell shape (Rauzi et al., 2008; Martin et al., 2009). In vitro, contact size is mainly determined by actomyosin contractility but not cadherin engagement (Maître et al., 2012). However, in Drosophila retina, N-cadherin mutants show drastic alteration of contact size and cell shape (Hayashi and Carthew, 2004), which suggests that cadherin-associated adhesion cannot be discounted. Even though the forces produced by cadherins and actomyosin networks act antagonistically, both systems are interconnected as cadherins are associated with intracellular actomyosin networks via catenins and other actin-binding proteins (Priya et al., 2013; Röper, 2015).

Due to the intrinsic links between cadherin-dependent adhesion and actomyosin contractility, it is challenging to address whether and how cadherin adhesion regulates cell shape. What is the direct contribution of cadherin-cadherin bonds to cell shape? Do cadherins influence cell shape through actomyosin contractility? To address these questions, we investigated the origin of cell shapes in vivo in the highly organized Drosophila retina, which features differential expression of cadherin molecules and is amenable to quantification of cell shapes and mechanical measurements. In particular, the Drosophila retina is an ideal system to study heterotypic contacts, and their differences with homotypic contacts.

Drosophila retina is composed of approximately 750 facets called ommatidia (Cagan and Ready, 1989; Tepass and Harris, 2007), each of which includes four cone cells (C) embedded in two primary pigment cells (P), along with other cell types shared by neighboring ommatidia (Figure 1A,B). The pattern of cone cells arrangement is strikingly similar to that of soap bubbles (Hayashi and Carthew, 2004). While this visual resemblance suggests that cells might minimize their surface of contact, both contractility and adhesion have to be considered for cell shape and cell arrangements (Lecuit and Lenne, 2007), as indicated by physical models (Käfer et al., 2007; Hilgenfeldt et al., 2008). Two classical Type I cadherins, E-cadherin (Ecad) and N-cadherin (Ncad) are expressed in the retina and specific expression of N-cadherin solely in cone cells governs the cone cell shape and arrangements (Hayashi and Carthew, 2004). In silico predictions based on energy minimization reproduce well the cone cell shapes but have limited experimental support (Käfer et al., 2007; Hilgenfeldt et al., 2008). In particular, the contributions of Ncad-mediated actomyosin contractility, as well as the interfacial tension in cone cell shape control, have not been explored.

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Patterns of *Drosophila* eye with the distributions of cadherins and Myosin-II (MyoII) in wildtype and *Ncad^M19* mosaic ommatidia.

(A) Image of pupal retina at 41 hr after puparium formation (APF) consisting of repeating lattice structure called ommatidia labeled with Ecad::GFP (green) and Ncad::mKate2 (red). (B) A schematic of the most apical view of an ommatidium, which contains four cone cells (C) and two primary pigment cells (P), and the localization of cadherins (Ecad in green and Ncad in red). (C–E) An individual ommatidium with Ncad::GFP in red (C), Ecad::GFP in green (D), Zip::YFP in magenta (E). (**F–G**) Wildtype and *Ncad^M19* mosaic ommatidia labelled with Ecad::GFP (green), Ncad (red) and Zip::YFP (magenta). *Ncad^M19* cone cells are marked by white asterisks. Magenta arrowheads in (F) shows the angle change in full *Ncad^M19* cone cells compared to wildtype. White arrowhead indicates the C|C contact with homophilic complexes and cyan arrowhead indicates the C|P contact in (C). Yellow arrowheads indicate one of the contacts at the interface between wildtype and *Ncad^M19* cells to highlight the absence of Ncad adhesion in (F) and significant increase in MyoII levels in (G). Scale bar, 10 µm.

https://doi.org/10.7554/eLife.22796.002

Ncad is involved in numerous morphogenetic processes including cell migration, neural tube formation, and axon guidance (Derycke and Bracke, 2004; Hirano and Takeichi, 2012; Lee et al., 2001). To date, the direct implication of Ncad and actomyosin complexes on cell sorting and patterning during development is unclear. Ncad depletion in Xenopus neural plate leads to the loss of activated form of myosin light chain (Nandadasa et al., 2009). Actin cytoskeleton remodelling in Drosophila glial cells is tightly regulated by Ncad levels (Kumar et al., 2015). In cell culture, a dynamic interaction was reported between Ncad and actomyosin complexes in myocytes (Comunale et al., 2007; Ladoux et al., 2010; Shih and Yamada, 2012; Chopra et al., 2011), neurons (Bard et al., 2008; Luccardini et al., 2013; Garcia et al., 2015; Okamura et al., 2004; Chazeau et al., 2015) and fibroblasts (Ouyang et al., 2013).

Here we combine mechanical measurements, quantitative microscopy and modelling to revisit the role of Ncad in cell shapes and cell arrangement. We show that Ncad bonds contribute two fold less than Myosin-II (MyoII) to interfacial tension, but that Ncad also affects localization and levels of MyoII, and thus cell shapes. We reveal that heterotypic interfaces between Ncad-expressing and non-Ncad-expressing cells accumulate MyoII more than homotypic interfaces, thereby stabilizing specific cell configurations. Our results emphasize the interplay between cadherins and actomyosin networks, which determines cell shape and cell arrangements during morphogenesis.

Results

Cadherins and Myosin-II distribution in pupal retinas

To visualize the patterns of cadherins in ommatidia, we analyzed their expression in Ncad::GFP (Figure 1C) and Ecad::GFP knock-in retinas (Figure 1D) (See Material and methods for details). As previously reported (Hayashi and Carthew, 2004), Ncad is localized at cone cell-cone cell contacts (C|C), where it forms homophilic complexes (Figure 1C, white arrowhead). Ncad is also found at low level at the junctions between cone cell and primary pigment cell (C|P) (Figure 1C, cyan arrowhead and Figure 1—figure supplement 1A). At C|P contacts, Ncad cannot form trans-homophilic bonds but cis-homophilic bonds, as it is expressed in cone cells but not in primary pigment cells. In addition, Ncad-Ecad trans-heterotypic bonds appear to be absent, as Ecad mutant cone cell loses contact from the neighbouring Ecad expressing primary pigment cell (Hayashi and Carthew, 2004). Ecad is present in all cell contacts albeit at different levels. Ecad concentration is lower at C|C relative to C|P and at primary pigment cell and primary pigment cell contacts (P|P) (Figure 1D). To visualize the pattern of MyoII, we imaged Myosin heavy chain (Zip)::YFP knock-in retinas (Figure 1E), and Myosin light chain (Sqh)::GFP flies driven by Sqh promotor in Sqh mutant background (Figure 1—figure supplement 1B). We also stained Zip::YFP or Sqh::GFP retinas with Phospho-Myosin-II light chain antibodies which labels active MyoII to check how well they correlate with each other (Figure 1—figure supplement 1C,D). As reported earlier (Warner and Longmore, 2009; Yashiro et al., 2014; Deng et al., 2015), Phospho-Myosin-II light chain antibodies show a punctate distribution, but overall the three markers indicate that MyoII is enriched at cell contacts and is also present as apical mesh at a lower concentration.

Loss of N-cadherin not only affects cone cell shape but also Myosin-II levels

To assess the impact of Ncad on cone cell shapes, we performed mosaic analysis to generate Ncad loss of function (Ncad^M19) clones in pupal retinas. Ncad mutation in one or multiple cone cells results in significant cell shape changes (Figure 1F), as reported earlier (Hayashi and Carthew, 2004). Shape variations are dependent on the numbers and combinations of wildtype and Ncad^M19 cone cells in the mosaic ommatidia (Figure 1F). In a full Ncad^M19 ommatidium, the four cone cells acquire a cruciform shape rather than the normal diamond shape (last and first image respectively in Figure 1F). Reduction in cell contact length (Figure 1F, yellow arrowhead) and change in angles (compare first and the last image of Figure 1F, magenta arrowhead) suggests that adhesion by homophilic bonding of Ncad causes a significant expansion of contacts between cone cells. Apart from the cell shape changes, there are variations in MyoII levels at mosaic Ncad^M19 ommatidium. For instance, at wildtype and Ncad^M19 cone cell contact, there is a significant increase in MyoII level (Figure 1G, yellow arrowhead). So, the loss of Ncad induces change in MyoII concentrations, suggesting a possible contribution of MyoII contractility in shaping cone cell patterns (Figure 1G).

Differential Myosin-II levels and interfacial tension

To explore the role of contractile forces in cone cell shapes, we determined the distribution of MyoII, a proxy for contractility, and measured interfacial tension acting at cell contacts in wildtype and Ncad^M19 mosaic ommatidia.

We used Zip::YFP fluorescence intensity as a readout of MyoII concentration. We observed different levels of MyoII at cell contacts, depending on whether (i) the two cells, for example cell 1 and cell 2 in contact express both Ecad and Ncad (1(E,N)|2(E,N)), (ii) the two cells in contact express only Ecad (1(E)|2(E)), (iii) one of the two cells in contact expresses only Ecad and another expresses both Ecad and Ncad (1(E)|2(E,N)) (Figure 1G, yellow arrowhead and Figure 1F,G).

In wildtype ommatidia, MyoII level was found 2.2-fold higher at the contact between cone cell and primary pigment cell, C(E,N)|P(E), than at C(E,N)|C(E,N) contacts. MyoII at contacts between primary pigment cells, P(E)|P(E), was found 1.8-fold higher than at C(E,N)|C(E,N) contacts (Figure 2A–C, Supplementary file 1 - table 1). A same trend in MyoII concentration is also observed when using Sqh::GFP as marker (Figure 2—figure supplement 1A–C, Supplementary file 1 -table 1). Interestingly, in Ncad^M19 mosaic ommatidium comprised of two Ncad^M19 cone cells, we again observed three distinct levels of MyoII depending on the genotype of the two cone cells in contact (WT and WT (C(E,N)|C(E,N)), WT and Ncad^M19 (C(E,N)|C(E)), Ncad^M19 and Ncad^M19 (C(E)|C(E))) (Figure 2D–F and Figure 2—figure supplement 1D–H, Supplementary file 1 -table 1). These data revealed a simple gradation in MyoII concentration c_Myo, similar to the wildtype at C(E,N)|C(E,N), P(E)|P(E) and C(E,N)|P(E) contacts: c_Myo(C(E,N)|C(E,N))=1, c_Myo(C(E)|C(E))=1.6, and c_Myo(C(E,N)|C(E))=2.3 (in arbitrary units). Our data indicates that differences in MyoII concentrations at contact are dependent on Ncad expression in the cells.

Figure 2 with 2 supplements see all

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Differential MyoII levels and interfacial tensions at various cell contacts.

(A) Wildtype ommatidium with Zip::YFP represented by (B) a schematic that highlights three different types of contacts at cell interfaces that express Ecad or Ncad or both E and Ncad. C(E,N)|C(E,N) contact (blue) shared by two cone cells, P(E)|P(E) contact (green) shared by two primary pigment cells and C(E,N)|P(E) contact (red) shared by a cone and a primary pigment cell. Scale bar, 10 µm. (C) Quantification of MyoII intensity in C(E,N)|C(E,N) (n = 30), P(E)|P(E) (n = 22) and C(E,N)|P(E) (n = 36) contacts. P-values are shown above the black horizontal lines (non-parametric Mann-Whitney U test on pairs and Bonferroni correction). (D) A *Ncad^M19* mosaic ommatidium with Zip::YFP. *Ncad^M19* cells are marked by white asterisks. Scale bar, 10 µm. (E) A schematic represents the corresponding *Ncad^M19* mosaic mutants highlighting C(E,N)|C(E,N) (blue), C(E)|C(E) (green) and C(E,N)|C(E) contacts (red). (F) Quantification of MyoII intensity in C(E,N)|C(E,N) (n = 30), C(E)|C(E) (n = 22) and C(E,N)|C(E) (n = 36) contacts. P-values are shown above the black horizontal lines. (G)-(K) Laser nanoablation experiments to estimate interfacial tension. (G) Schematic of a contact before (left) and after (right) ablation. Red cross represents the point of the ablation. Vertex A and B recoil changing distance 'd' after ablation. (H) Opening curve plots the distance’ d’ over time with a linear fit for initial time points to get the initial recoil speed. (I) Snapshot of an ablation at C(E,N)|C(E,N) contact in wildtype ommatidium, red cross indicates the ablation point. (J) Quantification of initial recoil speed of C(E,N)|C(E,N) (n = 14), P(E)|P(E) (n = 18) and C(E,N)|P(E) (n = 19) contacts in wildtype ommatidia. P-values are shown above the black horizontal lines. (K) Quantification of initial recoil speed in C(E,N)|C(E,N) (n = 14), C(E)|C(E) (n = 18) and C(E,N)|C(E) (n = 17) contacts in *Ncad^M19* mosaic mutants. Scale bar, 5 µm. P-values are shown above the black horizontal lines.

https://doi.org/10.7554/eLife.22796.005

Figure 2—source data 1 Dataset for Figure 2C,F,J and K.: https://doi.org/10.7554/eLife.22796.006
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Apart from MyoII, we also observed differences in Ecad levels when comparing C(E,N)|C(E,N), C(E)|C(E), C(E,N)|C(E) contacts (Figure 2—figure supplement 1F,G,H, Supplementary file 1 -table 2), raising the possibility that the changes in MyoII levels might be a consequence of Ecad homotypic interactions. However, MyoII levels are uncorrelated with Ecad levels, ruling out this possibility (Figure 2—figure supplement 1G and H).

MyoII level anti-correlates with cell contact length (Figure 2—figure supplement 1G,H), which is consistent with the idea that MyoII regulates length. One can argue that the knowledge of MyoII distribution is not sufficient to characterize contractility and that F-actin distribution and organization might also be an important determinant (Reymann et al., 2012). Thus, we stained for F-actin using phalloidin and found that F-actin is mostly apical and junctional like MyoII, but its distribution does not strictly correlate with that of MyoII; homotypic C(E,N)|C(E,N) contacts show higher F-actin level than C(E,N)|P(E), C(E,N)|C(E), P(E)|P(E) and C(E)|C(E) contacts (Figure 2—figure supplement 2A–C).

In an attempt to determine the relationship between MyoII-dependent contractility and tensile forces at cell contacts, we performed laser nano-dissection experiments (Rauzi et al., 2008). The initial recoil speed after the cell contact ablation served as a proxy for interfacial tension (Figure 2G–I, Figure 2—figure supplement 2D, Videos 1 and 2). We found that tension at C(E,N)|C(E) contacts was the highest while tension at C(E,N)|C(E,N) contacts was the lowest (Figure 2J,K). These values correlate with the levels of MyoII (compare Figure 2C and J or Figure 2F and K) and are consistent with the hypothesis that MyoII is a major determinant of interfacial tension.

Video 1

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posterframe for video — Laser nano-ablation of C(E,N)|C(E,N) contact in wildtype ommatidium.

Ablation at 00:00:00. Frame rate is 1 s/frame. Labelling: β-cat::GFP. Scale bar, 5 μm.

https://doi.org/10.7554/eLife.22796.011

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Bound and unbound N-cadherin differentially impact on Myosin–II junctional localization

To determine whether and how Ncad might control cell shape through MyoII regulation, we focused on the links between Ncad and MyoII localization. We observed high level of Ncad at homotypic contacts (C(E,N)|C(E,N)) which also exhibit the lowest concentration of MyoII, by 1.8 fold lower than the P(E)|P(E) cell contacts. This suggests that homophilic Ncad at homotypic contact reduces MyoII levels (Figure 2A,C), in agreement with the idea that cadherin lowers interfacial tension at cell contacts (Maître and Heisenberg, 2013). At heterotypic contacts (C(E,N)|P(E)), where Ncad cannot form transhomophilic bond, Ncad was found at very low level (Figure 1C, Figure 1—figure supplement 1A) and MyoII at a higher level than at any other contact (Figure 2A,C). This suggests that unbound Ncad at heterotypic contact signals to MyoII and induces its accumulation. To confirm this hypothesis, we took advantage of the fact that the primary pigment cells do not express Ncad and asked if we could modify MyoII level at different cell contacts by Ncad misexpression.

Ncad misexpression in one of the primary pigment cell affected the shape of cone cells in contact with it (Figure 3A,B). Homophilic Ncad was detected at the C(E,N)|P(E,N+) contacts (Figure 3—figure supplement 1A and A’, yellow arrowhead) and MyoII levels at these modified contacts (C(E,N)|P(E,N+), Figure 3A,B, yellow arrowhead) were significantly reduced compared to wildtype C|P contacts (C(E,N)|P(E), Figure 3A,B, green arrowhead, Supplementary file 1 - table 1). This confirms our hypothesis that homophilic Ncad reduces MyoII level (Figure 3C). In addition, higher level of MyoII was detected at contacts between primary pigment cells with one of them misexpressing Ncad (P(E)|P(E,N+)) (Figure 3A,D red arrowhead) than at contacts between wildtype primary pigment cells expressing only Ecad (P(E)|P(E)) (Figure 3A,B,D, Supplementary file 1 -table 1).

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Misexpression of Ncad in primary pigment cells and MyoII accumulation and MyoII asymmetry at cell contacts.

(A) An ommatidium with Ncad misexpressed in one of the primary pigment cells (white +) with Zip::YFP in magenta. Green arrowhead indicates the C(E,N)|P(E) contact. Yellow and red arrowheads indicate the modified C(E,N)|P(E,N+) and P(E)|P(E,N+) contacts respectively. (B) A schematic of Ncad misexpression ommatidium with the modified C(E,N)|P(E,N+) (blue), wildtype C(E,N)|P(E) and modified P(E)|P(E,N+) (red) contacts. (C) Quantification of MyoII intensity in C(E,N)|P(E) (n = 20) and C(E,N)|P(E,N+) (n = 20) contacts. P-value is shown above the black horizontal line. (D) Quantification of MyoII intensity in P(E)|P(E) (n = 16) and P(E)|P(E,N+) (n = 16) contacts. P-value is shown above the black horizontal line. (E) Wildtype ommatidium with Ecad::GFP (green) and Sqh::Ch (magenta). (F) Schematic with a zoom-in of a C(E,N)|P(E) contact shared by cone cell and primary pigment cell representing the asymmetric distribution of MyoII. (G) Average linescan of Sqh::Ch (magenta) intensity with respect to Ecad::GFP intensity (green) normal to interfaces (n = 10 interfaces). (H) An ommatidium with Ncad misexpressed in one of the primary pigment cell (white +) with Sqh::Ch (magenta). White arrowhead indicates the modified P(E)|P(E,N+) contact. (I) Schematics with a zoom-in of a modified P(E)|P(E,N+) contact shared by primary pigment cell and Ncad misexpressed primary pigment cell representing the asymmetric distribution of MyoII. (J) Average linescan of Sqh::Ch intensity (magenta) with respect to Ecad::GFP intensity (green) (n = 13 interfaces). Scale bar 10 µm.

https://doi.org/10.7554/eLife.22796.013

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To test whether such property of Ncad is specific to the retinal epithelium or more general, we performed clonal misexpression of Ncad in the larval wing pouch which cells express only Ecad. We noticed higher level of MyoII at the boundary of clones compared to MyoII inside the clones or to the surrounding wildtype tissue (Figure 3—figure supplement 1B,C, cyan arrowheads). This indicates that MyoII regulation by Ncad is not specific to the retina.

At C(E,N)|P(E) contacts, Ncad is asymmetrically localized as it is expressed only in one of the two apposed cells. We thus wondered whether MyoII could also be asymmetrically localized. To address this, we measured the intensity profile of MyoII perpendicular to C(E,N)|P(E) contacts, using Ecad intensity as a marker for the contact position. Localization of Ecad::GFP, and thus the contact position, can be determined with a precision better than the diffraction limit given the high signal/noise ratio (5–22 nm) (Figure 3—figure supplement 2, See Materials and methods). We found that MyoII maximum intensity was systematically shifted towards the cell that expressed both Ecad and Ncad (Figure 3—figure supplement 2A,B). Importantly, the distance between MyoII and Ecad intensity peaks (Figure 3—figure supplement 2C) was found larger than the imprecision in peaks’ localization (Figure 3—figure supplement 2D, See Material and methods). This significant and systematic shift indicates that MyoII is enriched in the cortex of an Ecad- and Ncad-expressing cell when it is apposed to an Ecad-expressing cell (Figure 3E–G). Using Starry night (Stan), a membrane marker that has a higher signal/noise ratio than Ecad at C(E,N)|C(E,N) contacts (Figure 3—figure supplement 2E), we confirmed the asymmetry of MyoII at C(E,N)|P(E) contacts (Figure 3—figure supplement 2F,G). In contrast, we observed a symmetric localization of MyoII at C(E,N)|C(E,N) contacts (Figure 3—figure supplement 2F,H).

This increase in MyoII level is cell contact autonomous: we observed higher MyoII intensity at C(E,N)|P(E) contacts, irrespective of the other contacts of the cell (for instance, C(E,N)|C(E,N)). This increase is striking in Ncad^M19 mosaic ommatidia in which a single Ecad- and Ncad-expressing cell is surrounded by Ecad-expressing cells: we noticed an intense ring of MyoII at the cortex (Figure 3—figure supplement 3A,B,C, compare cells marked by white and green arrowheads, Figure 3—figure supplement 3C). To further confirm the above observation, Ncad was again misexpressed in primary pigment cells to check if it could induce MyoII asymmetry at the modified P(E)|P(E,N+) contacts. An asymmetric localization of MyoII in cells that express both Ecad and Ncad was observed at the P(E)|P(E,N+) contacts (Figure 3H–J).

To further explore how Ncad at heterotypic contacts could induce MyoII contractility, we expressed only the extracellular part of Ncad in one primary pigment cell (Figure 4A, white +). Such truncated Ncad can form adhesion bonds but cannot interact with the actomyosin network (Figure 4A, white arrowhead). We observed a change in contact shape and MyoII levels at the interface between the wildtype cone cell and primary pigment cell that misexpressed extracellular Ncad (Figure 4B–D, compare blue and red arrowheads, Supplementary file 1 -table 1), which confirms a role for homophilic Ncad bonds in the downregulation of MyoII contractility. However, MyoII levels at the contact between primary pigment cells, which included one cell that misexpressed extracellular Ncad showed no change in MyoII, when compared to full-length Ncad (Figure 4B,C,E, green arrowhead, Supplementary file 1 -table 1). This result suggested that cytoplasmic part of Ncad is required for the accumulation of MyoII at the C(E,N)|P(E) contacts.

Figure 4

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Cytoplasmic part of Ncad is required for MyoII accumulation in heterotypic contacts.

(**A–B**) An ommatidium misexpressing extracellular part of Ncad in one of the primary pigment cells (white +) with Ncad (A) and Zip::YFP (B). White arrowhead indicates the C(E,N)|P(E,ΔN+) cell contact with homophilic Ncad in (A), red arrowhead indicates C(E,N)|P(E,ΔN+) wildtype cell contact, blue arrowhead indicates modified C(E,N)|P(E) cell contact and green arrowhead indicates the unchanged P(E)|P(E,ΔN+) cell contact. (C) Schematic of ommatidium misexpressing extracellular part of Ncad shows the modified cell contacts, C(E,N)|P(E) contact (blue), wildtype C(E,N)|P(E) contact (red) and unaffected P(E)|P(E,ΔN+) contact (green). (D) Quantification of MyoII intensity in C(E,N)|P(E) (n = 28) and C(E,N)|P(E,ΔN+) (n = 28). P-value is shown above the black horizontal line. (E) Quantification of MyoII intensity in wildtype P(E)|P(E) (n = 19) and unaffected P(E)|P(E,ΔN+) contact (n = 19). Scale bar, 10 µm. P-value is shown above the black horizontal line.

https://doi.org/10.7554/eLife.22796.020

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The above data suggest that while homophilic Ncad reduces MyoII contractility at homotypic contacts, unbound Ncad is able to activate MyoII, and locally enhance contractility at heterotypic contacts through its cytoplasmic part.

Both local tension and cell-scale contractility determine ommatidia shape

To understand how tensions at cell contacts determine ommatidia shape, we sought to build a simple mechanical model integrating both local tension and cell-scale contractility. Following earlier works, we thus designed a 2D model based on the minimization of a tension-based energy function (Käfer et al., 2007; Hilgenfeldt et al., 2008; Farhadifar et al., 2007). Although retina is obviously a 3D system, we treat the plane of adherens junctions, where both adhesion and MyoII molecules are recruited, as a 2D system. Since retinal cells have a complex shape and are variant in the z-direction, the relevance of the model is therefore limited to the junctional plane. Such an energy-based model assumes that the system settles to a configuration of minimum potential energy, which is likely to be the case in vivo since the developmental process is very slow and quasi-static. We then assume that individual contacts have a local tension γ_loc. As shown by our experiments, γ_loc is likely to be determined by the concentration of MyoII and cadherins engaged at the contact. The contribution of γ_loc at each contact to the total energy of the system is simply γ_locl, where l is the contact length. In addition, and as shown by others (Käfer et al., 2007; Hilgenfeldt et al., 2008; Farhadifar et al., 2007), the contractile cortical network and the 3D cell volume constraint are likely to impose a 2D geometry constraint at the cell level. We encapsulate this in a perimeter elasticity term, in which deviations Δp of the cell perimeter p from a preferred cell perimeter $p_{0}$ yield an energy penalty $\frac{K}{2} \frac{(p - p_{0})^{2}}{p_{0}}$ . The elastic constant K, which we assume is the same for all cells, determines how big this penalty is. In two-dimension, the mechanical energy of the ommatidium thus writes:

E = \sum_{contact i j} γ_{l o c i j} l_{i j} + \sum_{cells i} \frac{K}{2} \frac{(p_{i} - p_{0 i})^{2}}{p_{0 i}}

While cell area can vary experimentally, in a range which is likely to be determined by volume constraint and cell elasticity, in the model we chose to fix the area using a Lagrange multiplier. This choice is driven by simplicity arguments. Unlike perimeter elasticity, area elasticity is not crucial to select a shape or configuration, but mostly set the cell size (Hilgenfeldt et al., 2008). Interfacial tension at a cell junction is, by definition, the derivative of the energy function with respect to junction length, and writes:

γ_{i j} = γ_{l o c i j} + K \frac{Δ p_{i}}{p_{0 i}} + K \frac{Δ p_{j}}{p_{0 j}}

Interfacial tension γ is thus the sum of the local term, $γ_{l o c}$ , and of a cell-scale elastic term, $γ_{e l} = K \frac{Δ p_{i}}{p_{0 i}} + K \frac{Δ p_{j}}{p_{0 j}}$ . Note that ablation experiments reveal the global interfacial tension γ.

The parameters of the model are the target perimeters, the local tensions, and K. We sought to determine as many parameters as possible from experiments. We reasoned that in the absence of forces applied by surrounding cells, cells should acquire their preferred (target) perimeter (Figure 5—figure supplement 1A). We thus performed circular ablations, separating a cell from all its neighboring cells to measure the target perimeter. After ablation, cells relaxed towards a circular shape in the plane of adherens junctions (Video 3). Note that the perimeter after relaxation was found to be typically 8% smaller (8.4 ± 1.2, n = 7) than prior to ablation (Figure 5—figure supplement 1B,C). In addition, laser ablation experiments in Figure 2 provided us with relative estimates of the interfacial tensions (γ) for the different contact types (C(E,N)|C(E,N), P(E)|P(E) and C(E,N)|P(E)). Note that all tensions (including K, which has the dimension of γ) were normalized by the interfacial tension measured for C(E,N)|C(E,N) contacts, and therefore are given in units of C(E,N)|C(E,N)=1. Using that $γ_{l o c} \approx γ - 2 K \frac{Δ p}{p_{o}}$ and having determined $\frac{Δ p}{p_{o}}, K$ is the only free parameter remaining in the model. To determine its value, we minimized the energy function using the Surface Evolver software starting from an unrealistic configuration (Figure 5—figure supplement 2A), until the equilibrium configuration was reached. We then fitted the resulting ommatidia shapes to experimental shapes using K as a fit parameter. To fit simulations to experimental geometries, we chose two geometrical descriptors: the angle formed by adjacent C|P contacts and the length ratio between two contacts (polar-equatorial (Lm) over polar-posterior (Ls) contacts) (Figure 5A). We simulated the wildtype and four different Ncad^M19 mosaic ommatidia, and applied a weighted least squares method to fit them altogether (Figure 5—figure supplement 2B). The best fit corresponds to K = 4.2 (in units of γ_{C(E,N)|C(E,N)}=1). The cell patterns obtained in silico for this value are in very good agreement with the cell patterns observed in vivo, for wildtype ommatidia and for Ncad^M19 mosaic ommatidia with different numbers and combinations of wildtype and Ncad^M19 cone cells (Figure 5B, Figure 5—figure supplement 2C,D). Interestingly, our estimate of K also indicates that elastic tension contributes to 1/3 to 1/2 of the total interfacial tension, depending on the cell contacts considered (Figure 5—figure supplement 2E).

Figure 5 with 4 supplements see all

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Simulations of cone cell shapes and contribution of cadherins and MyoII to cell shapes.

(A) Schematics of two axes of polarity, A-P and Eq-Pl, of cone cells (bottom) and fit parameters measured in experiments and simulations, contact angle between cone cell and primary pigment cell (θ), ratio of contact length shared by A/P and Eq/Pl cell (Ls) to contact length shared by Eq and Pl cells (Lm) (top). (B) Comparison of experimental images (lower panel) to the simulations (upper panel), *Ncad^M19* cells are marked by white asterisks. (C) Schematic of force balance resulting from adhesion of Ecad (*ω_E,* green) and Ncad (*ω_N*, red), MyoII dependent cortical tension at the cell contact (σ) and cortex elasticity due to actomyosin at the cell perimeter (*γ_el*) (both in magenta). (D) Relative contribution of MyoII dependent cortical tension (σ), Ecad adhesion (*w_E*) and Ncad adhesion (*w_N*) to the local tension term *γ_loc* for all contact types in wildtype and *Ncad^M19* mosaic mutants. (**E–E'**) Image of the ommatidium with (E) Eq and (E') Eq and Pl cone cells *Sqh^Ax3* mutant (white -). β-catenin staining in green. (**F–F'**) Image of the ommatidium with (F) Eq and (F') Eq and Pl cone cells expressing constitutively active form of Sqh, Sqh^T20ES20E (white +), β-catenin staining in red. Scale bar, 10 µm.

https://doi.org/10.7554/eLife.22796.022

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The balance of cortical tension and adhesion determines local tension

The rationale of the model presented above is to predict ommatidia shapes from tensions at the cell contacts measured by ablations, irrespective of MyoII or cadherin levels. Yet, local tension is likely to result from the balance between MyoII-dependent cortical tension and cadherin-based adhesion (Lecuit and Lenne, 2007), and we were interested in weighing their respective (direct) contributions. To do so, we measured concentrations of cadherin and MyoII molecules in different configurations for which we knew the local tension.

We assumed that adhesion molecules and motor molecules have an additive and antagonistic contribution to local tension (Maître et al., 2012). Hence, MyoII cortical tension σ is balanced by cadherin based adhesion ω, so that γ_loc= σ - ω (Figure 5C). At C(E,N)|C(E,N) contacts, both Ecad and Ncad contribute to the adhesion term, so that ω = ω_E + ω_N, while at C(E,N)|P(E) and P(E)|P(E) contacts, only Ecad contributes to adhesion, and ω = ω_E. For the sake of simplicity, we assumed that adhesion and MyoII-dependent cortical tension were proportional to the concentrations of cadherins and MyoII, respectively. It should be noted that whether MyoII molecules are recruited through an Ncad feedback or any other pathway is not relevant to how they contribute to local tension. Hence the feedback between MyoII and Ncad is not considered to estimate the respective contribution of cadherin and MyoII molecules to tension. From there, we could use the molecular concentrations (Figure 2C,F, Figure 5—figure supplement 3A,B and E,F) and local tensions γ_loc obtained from ablation experiments combined to numerical modelling (Figure 2J,K and Figure 5—figure supplement 2E) to infer the contributions of Ecad, Ncad and MyoII to the local tension of the different contact types (See Materials and methods). We found that MyoII has a very significant contribution to local tension, which is about two to five times higher than that of Ncad or Ecad depending on the contact type (Figure 5D). This data, in agreement with in vitro experiments on cell doublets (Maître et al., 2012), emphasizes the quantitative role of MyoII on cell shapes in vivo. It also indicates that control of cell shape by adhesion is mostly indirect, through the regulation of MyoII level by unbound Ncad. This is again exemplified by the higher contribution of MyoII to local tension in C(E,N)|P(E) and C(E,N)|C(E) contacts than in P(E)|P(E) and C(E)|C(E) contacts (Figure 5D, middle and bottom panels).

To confirm the importance of MyoII on cone cell shapes, we manipulated MyoII activity in cone cells. We first decreased MyoII contractility using Myosin-II light chain loss of function (Sqh^Ax3) mutant (Figure 5E,E’), and observed a massive increase in cell apical area in the mutant cells and change in cell contact length (Figure 5—figure supplement 4A Supplementary file 1 - table 2). Conversely, misexpression of constitutively active form of MyoII (Sqh^T20E.S21E) leads to a reduction of cell apical area (Figure 5F,F’) and change in cell contact length (Figure 5—figure supplement 4B). These changes in apical area suggest that shape changes resulting from MyoII loss of function or misexpression are dominated by cell-scale (apical MyoII) rather than cell contact-scale contribution of MyoII. This is exemplified by our simulations, in which a simple change of the (fixed) area yields a qualitatively good prediction of cone cell shapes in different mutant configurations (Figure 5—figure supplement 4, See Materials and methods). A more quantitative assessment on these experiments would most likely require additional terms of area elasticity. Note that to exemplify experimentally the contribution of MyoII to local tension, one would ideally want to selectively downregulate or upregulate MyoII at cell contacts only, which is technically very challenging.

Myosin-II localization mediated by N-cadherin regulates cell arrangement

Lastly, to test the relevance of our data for tissue morphogenesis, we analyzed ommatidia morphogenesis in wildtype and Ncad^M19 mosaic retinas, 21 hr after pupal formation (APF) for 5 and 9 hr, respectively (Figure 6A,B and Videos 4 and 5). Wildtype cone cells undergo stereotypic neighbor exchanges (Figure 6A). Anterior and posterior cells lose A-P contact, while equatorial and polar cells intercalate and form a new Eq-Pl contact (A-P to Eq-Pl contact transition) (Figure 5A). However, when imaging the Ncad^M19 mosaic mutants, we observed defects of this A-P to Eq-Pl transition. 98,2% of analyzed ommatidia where Ncad was mutated in either the equatorial or polar cell failed to transit (Figure 6B, red arrowheads, Figure 6C, n = 114). 100% of analyzed ommatidia where Ncad was mutated in both equatorial and polar cells failed to transit (Figure 6D, n = 16). We reasoned that this transition might be prevented due to the increase in tension at the transverse cell contacts where C(E,N)|C(E,N) contacts are transformed into C(E,N)|C(E) contacts, and indeed observed increased levels of MyoII in these contacts (Figure 6E,F). To further test this hypothesis, we estimated the energy of the system as a function of the central junction length in both vertical and horizontal configurations (Figure 6G,H). Note that this required to fix that length during the minimization process. We found that the model predicts an energy minimum in the vertical configuration in both cases (when either 1 or 2 of the polar and equatorial cells are Ncad mutants), consistent with our experimental observations. Thus, cell mechanical properties, indirectly controlled by Ncad expression, not only impact on cell shapes but also on cell arrangement.

Figure 6

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Ncad mediated MyoII contractility impacts on cone cell arrangements.

(A) Snapshots of a movie at different APF from wildtype retina labelled with β-cat::GFP. Scale bar, 5 µm. (B) Snapshots of a movie at different APF from *Ncad^M19* mosaic mutant with Ecad::GFP and *Ncad^M19* cells (red asterisks). Mosaic ommatidia that failed to undergo normal cell rearrangement are indicated by red arrowheads. Scale bar, 5 µm. (C), (E) Equatorial *Ncad^M19* cone cell (white asterisk) in mosaic mutant with Ecad::GFP (green) and Ncad (red) in (C) and Zip::YFP (magenta) in (E) (both (C) and (E) total n = 112). (D), (F) Image of equatorial and polar *Ncad^M19* cone cells (white asterisks) with Ecad::GFP (green) and Ncad (red) in (D) and Zip::YFP (magenta) in (F) (both (D) and (F) total n = 16). (G) Energy profile of ommatidia with an equatorial *Ncad^M19* cone cell as a function of the central contact length (left direction: vertical contact length, right direction: horizontal contact length). Diagrams show corresponding simulations, with occurrence numbers observed experimentally. (H) Energy profile of ommatidia with equatorial and polar *Ncad^M19* cone cells as a function of the central contact length (left direction: vertical contact length, right direction: horizontal contact length). Diagrams show corresponding simulations, with occurrence numbers observed experimentally.

https://doi.org/10.7554/eLife.22796.032

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Discussion

We showed that the adhesion provided by Ecad and Ncad homophilic bonds have a moderate direct contribution to interfacial tension as compared to MyoII dependent contractile forces. Our in vivo findings are consistent with in vitro measurements using the shapes of cell doublets to infer the relative contribution of adhesion and cortical tension to interfacial tension (Maître et al., 2012). Here we demonstrate that in vivo, the contribution of adhesion to interfacial tension is roughly half of MyoII cortical tension. Our data indicate that the hypotheses of differential contractility (Harris, 1976; Brodland, 2002) or differential adhesion (Steinberg, 1963) are not mutually exclusive, and the balance of contractility and adhesion determines cell shapes, cell arrangement (Lecuit and Lenne, 2007; Käfer et al., 2007; Hilgenfeldt et al., 2008) and cell sorting (Krieg et al., 2008). The moderate contribution of adhesion bonds to interfacial tension might explain why cadherin binding affinities are not predictive of cell sorting outcomes in vivo and in vitro (Shi et al., 2008; Leckband and Sivasankar, 2012).

Our work unravels a cell-scale (autonomous) and a junction-scale (non-autonomous) control of cell shape through actomyosin contractility. Following previous models of epithelial mechanics (Käfer et al., 2007; Hilgenfeldt et al., 2008), we confirm that actomyosin contractility generates a cell-scale elastic tension at the cell periphery, which restricts cell deformation. This elastic tension is likely to be dependent on the stiffness of the actomyosin network bound to the membrane (Salbreux et al., 2012). Our data constrain the model and reduce the number of free parameters down to one, an effective elastic constant. Our model shows that the cell-scale elasticity is crucial to stabilizing the four-cone cell arrangement and it is possible that cell elasticity also ensures correct global patterning of the retina. Analysis of our measurements of mechanical properties and quantification of molecular distribution demonstrate that MyoII contractility also contributes locally to tension at cell contacts (cortical tension) to shape cone cell arrangement. This local contribution of MyoII to tension was not considered in previous works (Käfer et al., 2007; Hilgenfeldt et al., 2008). The cell-scale elasticity and junction-scale cortical tension contributions are on the same order of magnitude (Figure 5—figure supplement 2E) and are both crucial to predicting cell shape.

MyoII distribution and thus contractility is strongly dependent on cadherins. While the role of Ecad on contractility during tissue morphogenesis is well documented (Lecuit and Yap, 2015), the role of Ncad is poorly known. We identified a dual role of Ncad on cell shapes and cell arrangement. Junctional N-cadherin bonds yield contact expansion between Ncad-expressing cells. However, this effect is moderate and cannot alone account for the shapes of cells in the ommatidia. Through the determination of MyoII distributions at cell contacts, we uncovered another mechanism mediated by Ncad at heterotypic cell contacts, where a low level of Ncad is detected at junctional plane (unbound). Heterotypic contacts between cells expressing Ecad and Ncad and cells expressing Ecad only exhibit increased local contractility as compared to homotypic contacts. This difference in contractility cannot be explained only by differences in adhesion contributed by both Ecad and Ncad. This is a junction-autonomous property, as in an Ecad- and Ncad-expressing cell (C), we observed increased contractility at heterotypic contacts irrespective of the other contacts of the cell (heterotypic and/or homotypic). Our data suggest that unbound Ncad has the ability to redirect MyoII at heterotypic contacts via its signaling intracellular region. Interestingly, this does not seem to be specific to the retina and might be a more general mechanism, as suggested by our observations in the larval wing disc. N-cadherin was found to polarize MyoII contractility directly through it cytoplasmic partners such as β-catenin (Ouyang et al., 2013) or indirectly through its interplay with Ecad (Scarpa et al., 2015), presumably through an indirect mechanism. Cadherin-mediated adhesion is tightly coupled to actomyosin through small GTPase including Rho and antagonistic Rac (Takeichi et al., 1997; Ratheesh et al., 2013). Homophilic N-cadherin dimerization activates Rho (Comunale et al., 2007; Charrasse et al., 2002; Marrs et al., 2009; Taulet et al., 2009; Puvirajesinghe et al., 2016) and Rac (Matsuda et al., 2006). Also, actin organisation has been shown to be able to affect MyoII (Reymann et al., 2012). We did not detect any significant variation in Rho activities among different contacts of the ommatidia using a biosensor which detects active Rho1 (Munjal et al., 2015)(data not shown). Further experiments will be required to resolve the mechanism by which unbound Ncad could activate MyoII.

High MyoII contractility induced by cell contact molecules at tissue boundary has a significant impact on tissue separation (Dahmann et al., 2011; Major and Irvine, 2006; Fagotto, 2014). In Drosophila, supracellular actomyosin structures are found at boundaries in wing imaginal discs (Major and Irvine, 2006; Landsberg et al., 2009; Monier et al., 2010; Umetsu et al., 2014; Bielmeier et al., 2016) and embryos (Monier et al., 2010; Röper, 2013; Laplante and Nilson, 2011). We show here that the four cone cells in ommatidia form a boundary with primary pigment cells through increased MyoII contractility at the C(E,N)|P(E) heterotypic contacts. This MyoII cable is reminiscent of that triggered by Crumbs anisotropy at the border of placodes in the Drosophila (Röper, 2012). Cells inside the placodes have higher levels of Crumbs than cells outside placodes. In the peripheral placode cells, Crumbs homophilic interactions, which are thought to negatively regulate MyoII, lead to the selective accumulation of the Myosin cable at the boundary depleted of Crumbs. One could envision that Ecad anisotropy could lead to the accumulation of MyoII at the cell contacts having a high level of Ecad. We ruled out this possibility here as we found conditions where MyoII and Ecad anisotropy do not correlate (Figure 2—figure supplement 1G). In the retina, we showed that accumulation of MyoII is junction-autonomous and determined by the expression of adhesive molecules in the apposed cells.

At the heterotypic contacts, MyoII is asymmetrically distributed: it is mainly localized at the cortex of the Ecad and Ncad expressing cells. A recent study on the localization of polarity proteins on either side of cell interfaces made a similar observation (Aigouy and Le Bivic, 2016). From a mechanical point of view, the asymmetry of MyoII is an interesting observation as it suggests that tension can be set and modified asymmetrically. As a consequence, shrinkage or extension of a junction might be driven unilaterally from one of the two apposed cells. So far mechanical models of epithelia, including ours, do not take asymmetry into account, a property which would be interesting to explore further in the future. The adhesion molecules that are engaged in trans-bonds at cell contacts are symmetric in the apposed membranes. Thus, they cannot be the direct cause of this asymmetry. Instead, our data suggest that asymmetrically distributed unbound Ncad could signal to MyoII and cause its asymmetry. While asymmetric localization is an essential feature of planar polarity components (Goodrich and Strutt, 2011), it is largely unexplored for other junction constituents. It will be important to determine whether cytoskeletal components and regulators and members of adhesion complexes, also show asymmetric localization.

High MyoII contractility at contacts between two cell types might represent a general mechanism, which could be important for lineage sorting and elimination of misspecified cells (Bielmeier et al., 2016). Given the importance of E- to N-cadherin switch in epithelial-mesenchyme transition (Wheelock et al., 2008), our findings may also have implications in other developmental processes.

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Patterns of Drosophila eye with the distributions of cadherins and Myosin-II (MyoII) in wildtype and NcadM19 mosaic ommatidia.

Differential MyoII levels and interfacial tensions at various cell contacts.

Figure 2—source data 1

Laser nano-ablation of C(E,N)|C(E,N) contact in wildtype ommatidium.

Laser nano-ablation of C(E,N)|C(E) contact in NcadM19 mosaic ommatidium with polar (Pl) and posterior (P) cone cells(see Figure 5A for cone cell axes of polarity) lacking Ncad.

Misexpression of Ncad in primary pigment cells and MyoII accumulation and MyoII asymmetry at cell contacts.

Figure 3—source data 1

Cytoplasmic part of Ncad is required for MyoII accumulation in heterotypic contacts.

Figure 4—source data 1

Simulations of cone cell shapes and contribution of cadherins and MyoII to cell shapes.

Laser nano-ablation of target perimeter measurement (Δp/po).

Ncad mediated MyoII contractility impacts on cone cell arrangements.

A-P to Eq-Pl transition in wildtype retina.

Defects in cell rearrangements in NcadM19 mosaic mutants.

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Eunice HoYee Chan

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Pruthvi Chavadimane Shivakumar

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Raphaël Clément

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Edith Laugier

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Pierre-François Lenne

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Patterns of Drosophila eye with the distributions of cadherins and Myosin-II (MyoII) in wildtype and Ncad^M19 mosaic ommatidia.

Laser nano-ablation of C(E,N)|C(E) contact in Ncad^M19 mosaic ommatidium with polar (Pl) and posterior (P) cone cells(see Figure 5A for cone cell axes of polarity) lacking Ncad.

Defects in cell rearrangements in Ncad^M19 mosaic mutants.