1. Planar Cell Polarity Controls Pancreatic Beta Cell Differentiation and Glucose Homeostasis 
Cedric Cortijo, Mathieu Gouzi, Fadel Tissir, Anne Grapin-Botton 
Cell Reports 
Volume 2, Issue 6, Pages (December 2012)
DOI: /j.celrep
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2. Cell Reports 2012 2, 1593-1606DOI: (10.1016/j.celrep.2012.10.016)
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3. Figure 1
Celsr1, Celsr2, and Celsr3 Are Expressed in the Epithelium of the Pancreas
(A–H) In situ hybridization detecting Celsr1–3 mRNA expression was performed on 8 μm sections at E11.5 (A–C) and E14.5 (E–G). Celsr1 (A) and Celsr3 (C) are detected at E11.5, but not Celsr2 (B). Celsr1 (E), Celsr2 (F), and Celsr3 (G) are expressed in the pancreatic epithelium at E14.5. Sense probes for Celsr1–3 exhibited no staining as shown for Celsr3 (D and H). (A–D) Dotted lines show the pancreatic bud. Scale bars, 25 μm (A–D) and 100 μm (E–H), n = 4.
See also Figure S1.
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4. Figure 2
The Planar Cell Polarity Protein VANGL1 Is Restricted to Progenitor Cells
Representative sections of E10.5–E18.5 WT pancreata immunostained against the indicated antigens (A–H).
(A) E10.5 pancreas section immunostained with antibodies against PDX1 (white), pancreatic progenitor marker, and VANGL1 (red). No reproducible VANGL1 signal is detected, only random background.
(B) E11.5 pancreas section immunostained with antibodies against PDX1, Mucin1 (white), apical marker, and VANGL1 (red). VANGL1 is enriched close to the apical surface of cells.
(C and D) E14.5–E18.5 pancreas sections stained with Dapi (blue) and antibodies against ECAD (red), epithelial marker, Mucin1 (green), and VANGL1 (white). VANGL1 is expressed in the ductal compartment but not in the exocrine compartment delimited by the dotted line, preferentially at the apicolateral surface of cells (arrowhead).
(E–H) E14.5 pancreas sections immunostained with antibodies against PDX1, NEUROG3, endocrine progenitor marker, Insulin (INS) or Glucagon (GCG) (green), and VANGL1 (white). PDX1+ and NEUROG3+ cells are VANGL1+, whereas INS+, GCG+ cells and the acinar compartment (dotted line) are VANGL1−. (E′–H′) Only VANGL1 (white) is shown. Scale bars, 10 μm.
(I) Localization of VANGL1 along the lateral membrane of cells. Measurement of VANGL1 intensity signal at E18.5 along the apicobasal axis of the membrane shows a predominant expression at the apicolateral side of the cell (n = 31). The limit between apical and basal side of the membrane is positioned at the middle of the absolute length of the membrane. The distance from the apical (Mucin1) to the basal (mesenchyme) is set to normalize between the different cells.
(J) Area under the curve of apical VANGL1 versus basal VANGL1 intensity (n = 31).
∗∗∗p < Bars show SDs and p values are calculated according to Mann-Whitney U test.
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5. Figure 3
Celsr3 Deletion Results in Decreased β Cell Population and Glucose Intolerance
(A and B) E14.5 WT and Celsr3 KO serial pancreas sections were stained with Dapi (blue), and antibodies against Insulin (INS; red) and Glucagon (GCG; green). Scale bar, 50 μm.
(C and D) INS/Dapi area was quantified at E14.5 and E18.5. In Celsr3 KO embryos, INS area is significantly reduced compared to WT (n = 4), ∗p < (D) E14.5 Celsr3 f/f and Celsr3 f/f; Cre were stained with Dapi (blue) and an antibody against Insulin. INS/Dapi area was quantified at E14.5. In Celsr3 f/f; Cre embryos, INS area is significantly decreased compared to WT (n = 4), ∗p < 0.05.
(E) Intraperitoneal glucose tolerance tests were performed on 4-month-old Celsr3 f/f and Celsr3 f/f; Cre mice, fasted for 16 hr. Glucose (2 g/kg) was administered at time 0. Each point represents the mean of nine mice. Basal fasting blood glucose levels are similar between the two groups of mice. The Celsr3 f/f; Cre mice exhibit significantly higher blood glucose levels. (n = 9), ∗∗p < 0.001, ∗∗∗p <
(F) Insulin secretion normalized to total Insulin content in Celsr3 f/f and Celsr3 f/f; Cre islets at the indicated glucose concentrations (n = 4 mice for each genotype). No differences are observed.
(G) INS/Dapi area was quantified at 4 months. In Celsr3 f/f; Cre adults, INS area is significantly decreased compared to WT (n = 4), ∗p < Bars show SDs and p values are calculated according to Mann-Whitney U test.
See also Figure S2.
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6. Figure 4 Celsr3 and Celsr2 Promote Endocrine Cell Differentiation
(A) qRT-PCR analysis on E14.5 WT and Celsr3 KO embryos shows an increase in the relative expression of Celsr2 (n = 4), ∗p < 0.05.
(B and C) Serial pancreas sections from E18.5 WT and Celsr2+3 DKO embryos (DKO) were stained with Dapi (blue) and antibodies against INS (red) and GCG (green). Scale bar, 100 μm.
(D) At E14.5 and E18.5, INS/Dapi area was quantified. In DKO embryos, INS area is decreased compared to WT (n = 4), ∗p < 0.05.
(E) At E14.5 and E18.5, GCG/Dapi area was quantified. In DKO embryos, GCG area is decreased compared to WT (n = 4), ∗p < 0.05.
(F–H) The Ghrelin+, Pancreatic Polypeptide+, and Somatostatin+ cells were quantified at E18.5. In DKO embryos, Ghrelin+, Pancreatic Polypeptide+, and Somatostatin+ cells are decreased compared to WT (n = 4), ∗p < 0.05.
Bars show SDs and p values are calculated according to Mann-Whitney U test. See also Figure S3.
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7. Figure 5
CELSR2+3 Function via PCP Signaling and Control Endocrine Differentiation Downstream of NEUROG3
(A–C) E14.5 WT, Celsr3 KO, and Celsr2+3 DKO serial pancreas sections were stained with Dapi (blue), and antibodies against ECAD (red), epithelial marker, Mucin1 (green), and VANGL1 (white). Scale bar, 50 μm. VANGL1 is reduced at the plasma membrane of Celsr3 KO and almost absent in Celsr2+3 DKO (n = 4).
(D) Western blot against phospho-JUN. Celsr3 and Celsr2+3 deletion resulted in lower PCP signaling level (n = 3 independent experiments, each on three pooled E14.5 pancreas per lane).
(E and F) Mean of the quantification of the band intensity for Celsr3 KO and Celsr2+3 DKO (n = 3). ∗p < Bars show SDs and p values are calculated according to t test, hypothesizing a standard normal distribution.
(G and H) Serial pancreas sections of E14.5 WT and Celsr3 KO were stained with Dapi (blue) EdU (red), proliferation marker, and INS (green). Scale bar, 50 μm.
(I) The percentage of Insulin+ cells that costained with EdU is not different between WT, Celsr3 KO, and Celsr 2+3 DKO embryos (n = 4).
(J and K) Serial pancreas sections of E14.5 WT and Celsr3 KO were stained with Dapi (blue) and antibodies against SOX9 (red) and NEUROG3 (green). Scale bar, 50 μm.
(L) Quantification of the number of NEUROG3+ cells in WT, Celsr3 KO, Celsr3 f/f; Cre, and Celsr 2+3 DKO embryos did not show any difference (n = 4).
(M and N) Serial pancreas sections of E14.5 WT and Celsr3 KO embryos were stained with Dapi (blue) and antibodies against INSM1 (green). Scale bar, 50 μm.
(O) Quantification of INSM1+ cells shows a significant decrease in Celsr3 f/f; Cre and Celsr2+3 DKO compared to the WT. (n = 4), ∗p < 0.05.
Bars show SDs and p values are calculated according to the Mann-Whitney U test. See also Figures S4 and S6.
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8. Figure 6 Fate of Endocrine Progenitor Cells in Celsr3 KO Pancreas
(A–F) E18.5 Neurog3-Cre; Rosa26-YFP mice harboring WT or Celsr3 KO alleles immunostained with antibodies against DBA-lectin, ductal marker (white), YFP (green), and Insulin/Glucagon, endocrine marker (red). Arrowheads show YFP+ cells that are DBA+.
(G) Number of cells that are YFP+ and DBA+, normalized on the total DBA+ cells in pancreas. In Celsr3 KO embryos we observed that 35% progeny of NEUROG3-expressing cells remain confined within the DBA+ epithelium cells compared to 15% in the WT (n = 3).
(H) Number of cells that are YFP+ and DBA+, normalized to the total YFP+ cells in pancreas (n = 3).
(I) Number of cells that are YFP+ and CPA+, normalized to the total YFP+ cells in pancreas (n = 3).
(J) Number of cells that are YFP+, normalized to the total Dapi+ cells in pancreas (n = 3). No difference was observed. Scale bar, 10 μm.
Bars show SDs and p values are calculated according to the t test.
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9. Figure 7
Celsr2+3 Control Beta Cell Differentiation via the JNK Signaling Pathway
(A–D) E14.5 pancreas section immunostained with antibodies against PDX1, NEUROG3, INS and GCG, CPA, and Jun. (A and A′) PDX1+ progenitors located in cords are JUN+. (B–D′) Most of the NEUROG3+ endocrine progenitors are JUN+, although at a lower level than neighboring PDX1+ cells, whereas INS+ and CGC+ cells (endocrine cells) and CPA+ cells (acinar cells) are JUN−. (A′) PDX1 only (red). (B′–D′) Only JUN (green). Scale bar, 10 μm.
(E–G) E12.5 WT pancreatic explants were treated with DMSO or with 5 μM SP600125, a JNK inhibitor, diluted in DMSO for 48 hr. Quantification of immunostained explants for Insulin, Glucagon, and PDX1. The number of INS+/PDX1+ cells is significantly decreased (n = 4), ∗p < 0.05.
(H and I) Rescue experiment treating E12.5 Celsr3 KO pancreatic explants with water or with 50 ng/ml of anisomycin, a JNK agonist, diluted in water for 48 hr. Quantification of immunostained explants for Insulin and PDX1. The number of PDX1+ cells is reduced by anisomycin. The number of INS+/PDX1+ cells is significantly increased (n = 4), ∗p < Quantifications of INS cells were normalized to PDX1 to measure a differentiation flux from progenitors in these experiments where PDX1 cell numbers were affected by one of the compounds. Although some PDX1+ cells also express Insulin, their relative number is negligible.
Bars show SDs and p values are calculated according to the Mann-Whitney U test. See also Figure S5.
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10. Figure S1
Celsr3 Is a Target of NEUROG3 in the Pancreas, Related to Figures 1 and 2
(A) qRT-PCR with Celsr3 primers on 10 pooled pancreatic buds at E11.5 from WT or Neurog3 KO background. Standard errors are indicated.
(B and C) E12.5 pancreas section immunolabeled with antibodies against PDX1 (red), E-cadherin (green) and VANGL1 (white). VANGL1 is expressed only in a subset of PDX1+ epithelial cells at this stage and is absent from the peripheral tips (dotted line).
(D and E) Adult pancreas section stained with Dapi (blue) and antibodies against Vangl1 (red), and E-cadherin (green).
(F–I) Immunohistochemistry performed after ISH on section detecting Celsr3 mRNA. Sections were stained for Dapi (blue) and antibodies against Insulin (red), Glucagon (green) and Amylase (white). Scale bars = 10 μm.
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11. Figure S2
Supplemental Characterization of Celsr3 KO and Celsr3 f/f; Cre, Related to Figure 3
(A and B) At E14.5 in Celsr3 KO, PDX1+- and Dapi+-areas are transiently increased compared to the WT, thus revealing increased pancreas progenitors (n = 4), ∗p < 0.05.
(C and D) At E11.5 and E18.5, PDX1+- and Dapi+-areas respectively are unchanged compared to the WT (n = 4).
(E) The GCG/Dapi+ area was quantified at E14.5 and E18.5 revealing the proportion of Glucagon-expressing cells over total pancreas. In Celsr3 KO embryos, the GCG+ area is unchanged compared to WT (n = 4).
(F–H) In Celsr3 KO embryos, Ghrelin+-, Pancreatic Polypeptide+- and Somatostatin+ cell numbers are unchanged compared to WT (n = 4).
(I and J) In Celsr3 f/f; Cre embryos, the SOX9+- and Dapi+ -areas are equivalent to the WT (n = 4).
(K) The GCG/Dapi area was quantified at E14.5 and did not exhibit any difference.
(L) qRT-PCR for Celsr3 mRNA shows a recombination rate of 70% in Celsr3 f/f; Cre. The primers used were designed to recognize the deleted region of Celsr3.
Bars show SDs and p values are calculated according to the Mann-Whitney U test.
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12. Figure S3
Celsr2 Is Not Required for Endocrine Cell Differentiation, Related to Figure 4
(A) At E14.5 in Celsr2+3 DKO, the Dapi+-area representing the total pancreas is equivalent to WT.
(B) In Celsr2 KO, the SOX9+-area representing pancreas progenitors is comparable to the WT (n = 4).
(C) Quantification of NKX2-2+ cells shows a significant decrease in Celsr2+3 DKO compared to the WT, (n = 4).
(D) At E14.5 in Celsr2 KO, the SOX9+-area is unchanged compared to WT.
(E) Quantification of the number of NEUROG3+ endocrine progenitors in Celsr2 KO embryos did not show any difference (n = 4).
(F) The INS/Dapi area was quantified at E14.5 and E18.5 revealing the proportion of Insulin-expressing cells over total pancreas. In Celsr2 KO embryos, Ins area is not reduced compared to WT (n = 4).
(G) The GCG/Dapi+ area was quantified at E14.5 and E18.5 in Celsr2 KO embryos and no difference is observed compared to WT (n = 4). Bars show SDs and p values are calculated according to the Mann-Whitney U test.
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13. Figure S4
Membranous FZD3 Is Reduced in Celsr2+3 DKO and Normal Junctions in Celsr2+3 DKO, Related to Figure 5
(A and B) E14.5 WT and Celsr2+3 DKO serial pancreas sections were stained with Dapi (blue), and antibodies against E-cadherin (red), epithelial marker, Mucin1 (green), and Frizzled3 (white). Scale bar = 50 μm. Frizzled3 is drastically reduced at the plasma membrane of Celsr2+3 DKO (n = 4).
(C and D) Thin-section electron micrograph of pancreatic progenitor cells from a WT and a Celsr2+3 DKO embryo at E16.5. No difference in the structure of the adherens junctions is detected between WT and DKO (arrowhead). Scale bar = 250 nm.
(E and F) E14.5 WT and Celsr2+3 DKO serial pancreas sections were stained with Dapi (blue), and antibodies against E-cadherin (red). Scale bar = 50 μm. No difference of E-cadherin staining is observed.
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14. Figure S5
The NEUROG3 Population and the Proliferation Are Not Affected by the Inhibition of JNK, Related to Figure 7
(A and B) E12.5 WT pancreatic explants were treated with DMSO or with 5 μM SP600125, a JNK inhibitor, diluted in DMSO for 48 hr. Quantification of immunostained explants for NEUROG3 did not show any difference (t test). Quantification of the total EdU+ cells did not reveal any difference in proliferation (t test). No Insulin+ cells were proliferating, (n = 3). Bars show SDs.
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15. Figure S6
The Structure and Occurrence of Cilia Are Normal in the DKO, Related to Figure 5
(A–H) Thin serial section electron micrographs of pancreatic progenitor cells from a WT and a Celsr2+3 DKO embryo at E16.5. No difference in the structure of the cilia is detected between WT and DKO (arrowhead). Scale bar = 250 nm.
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