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Apical restriction of the planar cell polarity component VANGL in pancreatic ducts is required to maintain epithelial integrity

Cell polarity is essential for the architecture and function of numerous epithelial tissues. Here we show how planar cell polarity (PCP), so far studied principally in flat epithelia, is deployed during the morphogenesis of a tubular organ. Using the mammalian pancreas as a model, we report that components of the core PCP pathway such as the transmembrane protein Van Gogh-like (VANGL), are progressively apically-restricted. VANGL expression becomes asymmetrically localized at the apical surface of ductal cells, revealing a planar polarization of the pancreatic duct. We further show that restricting VANGL to these discrete sites of expression is crucial for epithelial integrity. Expansion of expression on basolateral membranes of the progenitors leads to their death and extrusion from the epithelium, as previously observed for perturbations of apico-basal polarity. Using organoids and in vivo analyses, we show that cell elimination is induced by a decrease of Rock activity via Dishevelled.


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Establishment of polarity is essential for the function of many cell types. In epithelia two axes of polarity 27 have been characterized: 1) apico-basal polarity orients cells from the free surface or the lumen to the 28 basal lamina and 2) planar polarity coordinates polarization of cell structures or behaviors in the plane of 29 the tissue, orthogonal to the apico-basal axis. While visualizing the apico-basal axis is relatively easy, few 30 cell types present overtly polarized structures suitable for the study of planar polarity. Much of what is 31 known regarding planar cell polarity (PCP) has been gleaned from studies of flat monolayers in which 32 planar polarity coordinates the orientation of cell appendices such as trichomes in fly wings or 33 stereocilia bundles in the inner ear 1 . Here, we investigate its organization and function in more complex 34 epithelia, such as tubes. 35 A key feature of planar polarity is the asymmetric localization of core molecules of the pathway along 36 both the apical-basal polarity axis and the plane of the epithelium. An evolutionarily-conserved core 37 We have previously shown that the expression of VANGL1/2 is initiated in the pancreatic epithelium at 83 around E11 when apico-basal polarity is first established 9 . Its cellular localization becomes progressively 84 restricted to apical foci at cell junctions ( Fig. 1a,b, S1d). In order to determine whether VANGL1/2 is 85 asymmetrically expressed in the plane of the epithelium, we performed whole mount staining which 86 allowed us to visualize pancreatic ducts in three dimensions (3D) (Fig. 1e-g). Co-staining for Mucin1 and 87 β-Catenin, respectively marking apical and lateral membranes, revealed that ductal cells were 88 anisotropic, exhibiting elongated apical surfaces along the longitudinal axis of the ducts (Fig. 1f,g). 89 VANGL1/2 was distributed asymmetrically in the apical plane, the protein being enriched on the 90 transverse membranes (perpendicular to the longitudinal axis of the duct) while it was almost absent on 91 the longitudinal membranes (Fig. 1f,h and movie 1). While an asymmetric expression pattern 92 characterizes the localization of PCP core components in flat monolayered epithelia, these findings 93 reveal that this is conserved in a branched, tubular network. We also observed an enrichment of 94 VANGL1/2 protein at tricellular junctions, "hot-spots" of epithelial tension 20 (Fig. 1g). 95 We then investigated the expression of Frizzled proteins which is also planar polarized in flat epithelia. 96 Although both FZD3 and FZD6 receptors can mediate PCP signaling 21 , we were only able to detect FZD3. 97 Similarly to VANGL1/2 expression, at E10.5, small foci of FZD3 protein were detected around forming 98 micro-lumens (Fig. S1a). At E12.5, FZD3 expression was enriched apically in the branching epithelium 99 (Fig. S1b). Though largely restricted to the membrane, some cytoplasmic expression was visible (Fig.  100 S1b). By E14.5, FZD3 was restricted to the apical-most regions of the lateral membranes, appearing as 101 sub-apical striations of expression (Fig. S1c), which, by E18.5, condensed to foci at the apical cell 102 junctions (Fig. 1d). Both FZD3 and VANGL1/2 were expressed in distal tip and proximal trunk cells of the 103 were subsequently detected in islets of Langerhans at E18.5 (data not shown). Taken together, the 105 membranous proteins of the PCP display progressive apical restriction in tubes as well as planar 106 expression for at least VANGL. 107 Ectopic expression of VANGL2 on baso-lateral membranes leads to cell death and pancreatic 108 hypoplasia 109 Though the importance of asymmetric expression of PCP proteins in the plane is well established in both 110 Drosophila 22 and vertebrates 23 , little is known regarding their apical restriction. To test the significance 111 of VANGL2 expression and localization, we generated transgenic mouse lines in which Cre-mediated 112 recombination inactivates β-actin promoter-driven GFP expression and induces expression of a Cherry-113 VANGL2 fusion protein (Fig. 2d). This construct was previously reported to express a functional 114 protein 24 . When expressed in pancreatic progenitors using Pdx1-Cre, the fusion protein was initially 115 detected at the cell cortex in of most pancreatic epithelial cells at E10.5 and by E12.5 became 116 membrane-enriched ( Fig. S2a-b). Mesenchymal cells surrounding the epithelium expressed GFP, as 117 expected from the non-recombined transgene (Fig. S2c). In contrast to endogenous VANGL proteins 118 which are restricted to the apical junction in wild-type pancreata (Fig. 2a), expression of the Cherry-119 VANGL2 fusion protein showed no such delimitation and was detected throughout the membrane (Fig.  120 2b-c). 121 To determine whether this ectopic expression of VANGL protein throughout the cell membrane 122 impacted upon pancreas development, we analyzed pancreatic epithelial size in double-transgenic Pdx1-123 Cre; Cherry-Vangl2 (Ch-Vgl2) mice versus control (single-transgenic) littermates. At E10.5, when Cherry-124 VANGL2 protein was first detectable in double-transgenic animals, their pancreata were equivalent in 125 size to controls (Fig. S2d) but by E12.5, we observed a ~60% decrease in Ch-Vgl2 pancreatic epithelial 126 individuals. 133 To test whether hypoplasia of Cherry-VANGL2-expressing pancreata resulted from reduced 134 proliferation, we quantified the proportions of phospho-HistoneH3-expressing cells in the epithelium. 135 No significant difference was detected between the ratios of proliferating epithelial progenitors in E12.5 136 Ch-Vgl2 versus control siblings ( Fig. S2g-h). In contrast, while TUNEL assays performed on E12.5 litters 137 revealed only a few (0.07% in average) apoptotic cells in control pancreatic epithelia, we observed a 2.2-138 fold increase in TUNEL + pancreatic epithelial cells in Ch-Vgl2 littermates (Fig. 2h). Moreover, the extent 139 of hypoplasia positively correlated with the proportion of apoptotic cells (Pearson correlation test: 140 r=0.8; p<0.05) (Fig. S2k). In the Ch-Vgl2 embryos, we also observed elevated TUNEL signal among the 141 mesenchymal cells located just in the periphery of the epithelium (Fig. 2h, arrow and Fig. S2j). We 142 presume that these cells, located at a distance of one or two cell diameters from the epithelium, are 143 epithelial cells that have recombined and extruded from the distal-most epithelial layer. Staining for 144 Caspase3 confirmed these observations, revealing an increased number of Caspase3 + cells in the 145 epithelium expressing the Cherry-VANGL2 fusion protein (Fig. 2i,j), with some of those cells departing 146 the outer-most epithelium (Fig. 2l). Furthermore, we detected dying (Caspase3 + ) cells retaining Cherry 147 expression, suggesting that Cherry-VANGL2 expression cell-autonomously manifests in cell death (Fig.  148 2k). Taken together, these data show that ectopic expression of Cherry-VANGL2 protein in the 149

Epithelial exit and destruction of epithelial integrity upon VANGL mislocalization to all cell membranes 152
To test the hypothesis that VANGL extension to all cell membranes may promotes epithelial exit, we 153 conducted live imaging on pancreatospheres. These structures comprise a spherical monolayered 154 pancreatic progenitor epithelium and are thus simpler than the branched pancreatic epithelium, and 155 more amenable for observation of epithelial exit in live imaging 25,26 . We generated pancreatospheres 156 from Ch-Vgl2 and control littermate pancreatic epithelium at E13.5, after PCP was established. We 157 consistently observed increased cell delamination in spheres originating from Ch-Vgl2 pancreata (movies 158 2, 3). These events lead to a disruption of the epithelium, resulting in inward collapse of 159 pancreatospheres (Fig. 2m, n and movies 4, 5, 6). We have recently shown that pancreatic progenitors 160 lining ducts secrete fluid into the lumen from E10.5 6 . We interpret that disruption of epithelium integrity 161 leads to collapse because of either a relase of internal fluid pressure or disruption of supracellular apical 162 cortical tension. 163 VANGL misexpression leads to decreased actomyosin contractility 164 Actomyosin contraction, mediated by activation of Rho kinase, is well-recognised as an important 165 downstream effector of the PCP 11,27,28 and VANGL, in particular, has been involved in remodeling of actin 166 microfilaments 29-33 . Moreover, actomyosin contractility is associated with apoptosis in different 167 contexts 34 . For these reasons, we examined phosphorylation of myosin regulatory light chain (pMLC), a 168 substrate of Rho kinase and effector/read-out of actomyosin-based contraction 35 in pancreata from Ch-169 Vgl2. Immunostaining showed pMLC to be apically polarized in E12.5 control pancreata while the 170 protein was undetectable in Ch-Vgl2 pancreatic epithelia ( Fig. S3a-b). At E14.5, pMLC was expressed at 171 the apical cell junction in controls (Fig. 3a,c), mirroring the distribution of core PCP components, but 172 following ectopic VANGL expression. 177 Jun N terminal Kinase (JNK) has been reported as a downstream effector of the PCP pathway although 178 its function downstream of the core components and especially of VANGL protein remains unclear 36 . 179 Western blots for phospho-c-jun did not reveal any significant dysregulation of the JNK pathway activity 180 in E14.5 Ch-Vgl2 compared with control pancreata (Fig. S3d-e). This suggests that the mechanism of 181 VANGL misexpression-induced cell death is independent of the JNK pathway. 182

RhoA pathway activation rescues cell death and pancreatic hypoplasia induced by VANGL 183 misexpression 184
So far, our analysis showed an increase of cell death and a decrease of ROCK pathway activity following 185 the misexpression of VANGL protein. In order to determine whether the decrease of ROCK pathway 186 activity was responsible for apoptosis in Ch-Vgl2 pancreata, we tested whether pharmacological 187 activation of the ROCK pathway in Ch-Vgl2 pancreatic epithelium is sufficient to rescue apoptosis and 188 hypoplasia. To manipulate the ROCK pathway conveniently, we used an ex vivo culture system similar to 189 our previously-published pancreatic organoid model 25 , but in which the whole E10.5 pancreatic 190 epithelium was seeded in Matrigel enabling growth in 3D in the absence of mesenchyme. The growth of 191 the developing pancreas was evaluated every day by measuring the circumference of the epithelial bud. 192 The pancreatic buds expressing Cherry-VANGL2 protein grew slower and remained smaller than those 193 from control littermates at the end of the culture period (five days), mimicking the phenotype observed 194 in vivo (Fig. 4a,b). Inactivation of ROCK activity (via H1152, 5 µM) in wild-type epithelium manifested in 195 we also observed a delay in branching of the pancreatic epithelium when the ROCK pathway was 197 inhibited ( Fig. 4c; asterisk). On the contrary, wild-type buds treated with a RhoA activator (CN03, 1 198 µg/ul) grew more rapidly and branched precociously compared with control vehicle-treated buds (Fig.  199 4e,f). Finally, activation of RhoA in Ch-Vgl2 pancreatic epithelium rescued the hypoplasia (Fig. 4g,h, 200 normalized data; for details, see Materials & Methods). In order to assess the level of activity of the 201 ROCK pathway resulting from the various perturbations above, we performed Western blotting for 202 pMLC on individual treated buds (Fig. 4i). While the amount of pMLC protein was decreased by ~70% in 203 untreated Ch-Vgl2 buds (70 ± 4%) and wild-type buds treated with ROCK inhibitor (68 ± 7%), in Ch-Vgl2 204 buds treated with RhoA activator, pMLC levels were comparable to controls, indicating that ROCK 205 pathway activity was restored to endogenous levels ( Fig. 4i). Lastly, we observed that Ch-Vgl2 buds as 206 well as those treated with ROCK inhibitor H1152 exhibited extensive apoptosis ( Fig. 4j-l). In combination, 207 these ex vivo experiments demonstrate that the cell death and pancreatic hypoplasia resulting from 208 VANGL2 misexpression is a consequence of decreased ROCK pathway activity. Intracellularly, Prickle directly binds to VANGL and can bind to Dishevelled to antagonize its Frizzled-221 mediated membrane recruitment 37-40 . Like VANGL1/2 and Frizzled3, Prickle2 is expressed at the apical 222 junctions in the pancreatic epithelium ( Fig. S4a-b). In Ch-Vgl2, we did not detect any changes in the 223 pattern of expression of Prickle2 at E13.5 or E14.5 (Fig. 5e-h and data not shown). 224 Dishevelled can be physically bound by VANG in Drosophila, and in vertebrates, these interactions are 225 believed to antagonize the formation of the FZD-DVL complex by affecting DSH/DVL levels and/or 226 stability 28,37,41,42 . In the E12.5 pancreatic epithelium, DVL2 protein is enriched apically in the duct and 227 becomes cytoplasmic in the newly-formed endocrine clusters (Fig. S4c). This expression pattern is 228 maintained at E15.5 ( Fig. S4d-g). High resolution expression analysis at this stage showed that contrary 229 to the other core components, DVL2 protein is not tightly restricted to the apical junction ( Dishevelled protein is not observed. Neither increased cell death, epithelium integrity defects nor ROCK 298 activity dysregulation were detected. Therefore, it appears that the ectopic localization of VANGL on the 299 basolateral membrane is the key determinant in the mechanism we propose and that restriction of 300 VANGL protein activity to the apical membrane is essential for cell survival. 301 Interactions between apico-basal polarity and planar polarity have been reported 11,59 but, so far, only 302 apico-basal polarity defects have been associated with apoptosis. In the fly it is well established that 303 disruption of apico-basal components such as crumbs, stardust or bazooka lead to apoptosis 60-62 . Our 304 work hereby reveals that disruption of a PCP core component expression domain can also manifest in 305 cell death. The downstream pathways involved differ as in fly, activation of the JNK pathway triggers 306 apoptosis 63 while we show that a decrease of ROCK pathway activity induces apoptosis following ectopic 307 expression of VANGL. ROCK signaling can serve as either a pro-apoptotic or pro-survival cue in a cell type 308 and context-dependent manner 64  Other stainings performed in whole-mount did not require any amplification, the samples were 392 therefore dehydrated stepwise in MeOH at this step. Samples stained with VANGL antibody were 393 washed again in TBST before incubation with Cy3 for 1h at RT (Perking Elmer TSA kit). Then, after 394 washing in TBST, the specimen were dehydrated in MeOH and stored at −20°C. All samples were cleared 395 in a solution of 1:2 Benzyle Alcohol and Benzyle Benzoate (BABB) for 12-24 hours prior to imaging. 396 Cleared specimens were subsequently mounted in glass concavity slides in BABB. Imaging was 397 performed using a Leica SP8 confocal microscope with a 20x/0.75 IMM CORR objective and Hybrid 398 detectors at 1024 × 1024 resolution in an 8-bit format. Three-dimensional reconstructions and movies 399 were performed with the Imaris software (Bitplane). 400

Immunohistochemistry on sections 401
18 piece and fixed in 4% PFA for 30min (E10.5 and E12.5), 1h (E14.5) or 2h (E18.5) at RT. E13.5 and E15.5 403 samples prepared for DVL2 staining were fixed 30 min in PFA diluted to 4% in water rather than the 404 usual PBS. After several washes in PBS, samples were equilibrated in 15% sucrose/PBS 0.12 M solution 405 overnight at 4°C prior to embedding in gelatin (7.5% gelatin diluted in 15% sucrose/PBS 0.12 M 406 solution). Gelatin blocks were subsequently frozen at -65°C in 2-methylbutane (isopentane, Acros 407 Organics) and kept at -80°C until sectioning. Frozen sections (8 to 10 µm) were then dried at room 408 temperature and washed in PBS Triton 0.1%. The slides were incubated 10min in PBS Triton 0.3% to 409 permeabilize the tissue. If necessary, an antigen retrieval step was performed at this point (see below). 410 If secondary antibodies directly conjugated to HorseRadish Peroxidase (HRP) were used, a 7 min 411 incubation in 3% H2O2 was performed at this point. The tissues were rinsed and then blocked 1h at RT in 412 the appropriate blocking reagent (Table 1, (Table 1, supp. Methods). The samples were brought back gradually to RT before proceeding to the next 428 step. For Frizzled3 staining we used a cycle of 20min at 95˚C. 429 TUNEL assays were performed with an ApopTag Peroxidase in situ apoptosis detection kit (Millipore, 430 S7100) and followed by immunofluorescence staining as described above. 431

Statistical analysis 486
Statistical analyses were performed with GraphPad 6 software packages and Microsoft Excel. 487 Normalization to control littermates was obtain by making a ratio between the value obtained in the 488 transgenics (or mutants) and the average value of controls present in the same litter, multiplied by 100 489 to convert to percentages. All statistical tests in the paper are unpaired non-parametric student t-tests. 490 Normality of the data cannot be tested, we thus assume that the data have a Gaussian distribution. representative area of whole mount imunotaining illustrating delamination of a CAS3+ cell. m-n, pancreatospheres generated from control or Ch-Vgl2 pancreata were imaged 4 days after seeding for either 3 hours (litter1) or 5 hours (litter2). m, Phenotypes were scored at the end of the movie: complete loss of lumen (collapsed), reduction of lumen size (collapsing), lumen diameter unchanged or increased (normal). A Chi-square test shows a significant difference between spheres generated from control or Ch-Vgl2 with respect to the phenotype 'reduced lumen size' (collapsing + collapsed). Chi-square value = 52.6, p-value < 0.00001. n, Snapshots of representative spheres at the beginning and end of the movie. Additional examples of collapsing spheres are shown in movie 2-7. Scale bar 5 µm (a,b); 10 µm (c,h-l,n); 50 µm (f-g)  Apical overexpression of VANGL protein due to Inversin mutation does not lead to the perturbation of actomyosin contractility nor cell death. a-d, longitudinal (a, c) and transversal (b, d) sections of E18.5 pancreatic duct immunolabelled for VANGL (magenta) show an increase of the protein at the apical membrane in the inversin mutant (c, d) compared to the WT littermate (a, b). e, quantification of the epithelium area in the inversin mutant and the control littermates at E12.5 and E14.5 shows no difference in the size of the epithelium. p-value>0.5 by t-test. f, quantification of the number of Caspase3 positive (CAS3+) cells in E12.5 inversin mutant shows no significant difference in apoptotic cells compared to the control. The CAS3+ cells/ epithelium area ratio was normalized against the control littermates. p-value>0.5 by t-test. g-h, staining for pMLC in E14.5 WT and Inversin pancreatic sections shows no differences in the localization of the protein. i, representative image of a western blot against pMLC. Quantification of the pMLC/Tubulin ratio shows no difference between inversin homozygote, heterozygote mutant and their control littermates. p-value>0.1, by t-test. j-k, Z-projection of confocal images showing Dishevelled2 (DVL2) expression on E12.5 pancreatic sections. No differences of localization or level of DVL2 were observed between the control (j) and the inversin mutant (k). *, highlighted endocrine islet. Scale bar 10 µm.