Nandadasa S , Tao Q , Menon NR , Heasman J , Wylie C .

R01-HD044764 NICHD NIH HHS , R01 HD044764 NICHD NIH HHS

	Fig. 1. Temporal and spatial expression patterns of classical E-, C- and N-cadherins. (A) Real-time RT-PCR analysis of cDNA isolated from Xenopus embryos at different stages showing the temporal expression pattern of N- (red), E- (blue) and C- (yellow) cadherin. Green bars show levels of the loading control, ODC. (B) E-cadherin is expressed at st. 11 in animal cells at low levels (left panel), is lost from the dorsal (neural) ectoderm, but is expressed strongly in the ventral (non-neural) ectoderm at st. 13 (center two panels), and is expressed primarily in the lateral and basal regions of ventral ectoderm cells (right-hand panel). (C) C-cadherin is expressed in both neural and non-neural ectoderm of the neurula (left two panels), and is expressed primarily in the lateral and basal regions of the neural plate, but in the apical regions of the non-neural ectoderm (right-hand two panels). (D) N-cadherin is expressed in the neural, but not non-neural, ectoderm at st. 13 (left panel), primarily in the apical cytoplasm (right-hand panel). Dashed line demarcates junction between neural and non-neural ectoderm. (E) The distribution of N-cadherin in sections of a st. 40 embryo. White brackets in B-D indicate the ectoderm layer in each cross-section. CNS, central nervous system; HT, heart; N, notochord. Scale bars: 50 μm, except 200 μm in C (left) and 100 μm in C (right), D (left) and E.
	Fig. 2. E- and N-cadherin can replace C-cadherin to assemble cortical actin during the blastula stage. (A) Cortical F-actin levels in cells of the inner surfaces of dissected Xenopus animal caps from embryos that were untreated (Control), depleted of maternal C-cadherin mRNA (C-cad AS) or depleted of C-cadherin mRNA and subsequently injected with either HA-tagged E-cadherin mRNA (C-cad AS+Xl-E-cad HA) or Myc-tagged N-cadherin mRNA (C-cad AS+Xt-N-cad Myc). Expression of either E- or N-cadherin rescues the loss of cortical actin caused by C-cadherin depletion. (B) Cadherin levels in animal caps from the same experiment. The left-hand panels show the degree of depletion of C-cadherin protein (compare Control with C-cad AS, stained with anti-C-cadherin antibody). The right-hand panels show that both E-cadherin and N-cadherin, stained using anti-HA and anti-Myc, respectively, were expressed at the cell surface. (C) Mean F-actin pixel intensity quantified for each group shown in A. ***Statistically significant difference between control and C-cad AS levels of phalloidin staining (P<0.001). Scale bars: 50 μm.
	Fig. 3. N-cadherin is required to assemble F-actin in the apical cytoplasm of neural plate cells. (A-D) Injection of a 16-cell embryo (A; the arrow indicates the injected cell) gives rise to labeled clones in the neural plate at st. 14 (B; the dashed line indicates boundaries of the neural plate). Neural plates (C) were stained for F-actin (D). (E) The boundary between N-cadherin-depleted (*) and uninjected (#) cells in a neural plate stained with N-cadherin antibody to show the reduction in N-cadherin protein in the injected cells. (F) Transverse section of a neural plate showing that C-cadherin continues to be expressed in N-cadherin-depleted embryos. (G) The dramatic reduction in F-actin in N-cadherin-depleted cells (red) as compared with untreated neighboring cells of a neural plate. (H) Quantitation of staining intensity for F-actin and of mean apical surface area of injected as compared with untreated cells. (I) Transverse section through the neural plate in G, to show that only the apical regions of the neural plate cells are affected by N-cadherin depletion. White arrows indicate circumferential cortical actin belt. Yellow arrows indicate F-actin assembly is retained in the basal cytoplasm of N-cadherin-depleted neural plate cells. (J) Injection of Xenopus tropicalis N-cadherin mRNA, which does not include sequence complementary to the MO, rescues the F-actin expression in N-cadherin-depleted cells. The left-hand panel shows the RLDX lineage tracer (red); the center panel shows only the F-actin staining in the same specimen. Asterisk indicates the treated cells; #, the untreated region. The right-hand panel shows quantitation of the cortical actin staining from the pixel intensity. ***Statistically significant difference in phalloidin staining between control and N-cadherin-depleted neural plate cells (P<0.001). Scale bars: 50μ m, except 100 μm in J and 200 μm in D.
	Fig. 4. N-cadherin depletion in the neural plate blocks neural fold closure and causes spina bifida. (A) The two dorsal animal cells injected with anti-N-cadherin MO (arrows), and the clone of cells derived from this at st. 13 (right-hand panel). (B) Frames from a movie showing failure of folding of the neural plate in MO-injected Xenopus embryos (right-hand panels) as compared with untreated embryos (left-hand panels). The arrow indicates a distinct lip in the neural plate (NP) caused by the pushing movement of the non-neural ectoderm (NE). (C) Spina bifida in N-cadherin-depleted as compared with untreated embryos. N-AS, N-cadherin-depleted embryos; U, untreated embryos.
	Fig. 5. N-cadherin depletion causes loss of activated myosin light chain in the apical cytoplasm in neural plate cells. (A,B) En face and transverse sectional views, respectively, of an untreated Xenopus embryo, stained at st. 16 with anti-phosphorylated myosin light chain antibody. Staining is intense in the apical cytoplasm of the center of the folding neural plate. (C,D) Corresponding views of an embryo with a clone of N-cadherin-depleted cells in the neural plate; in the region of the clone (asterisk with bracket), staining is lost in the apical cytoplasm of the neural plate cells. Scale bars: 200 μm in A,C; 50 μm in B,D.
	Fig. 6. Depletion of E-cadherin in the ventral (non-neural) ectoderm causes reduction of F-actin. (A,B) Injection of E-cadherin MO into single ventral animal cells (arrow in A) gives rise to large clones of cells depleted of E-cadherin protein (lower panel in A, B). Arrows in B indicate boundaries between injected cells (*) and uninjected cells (#), which express E-cadherin. (C) Reduction of F-actin in E-cadherin-depleted cells (*) as compared with uninjected cells (#). (D,E) Reduction, but not absence, of F-actin in E-cadherin-depleted cells (D, lower panel), probably because of continued expression of C-cadherin, as shown en face and in cross-section in E. (F) Most of the non-neural ectoderm is labeled by injection of both ventral animal cells at the 8-cell stage. The same embryo is shown in the center and lower panels during neural fold closure, which is delayed compared with untreated embryo (left) owing to reduced pushing movements of the non-neural ectoderm (NE). (G) Rescue of the cortical actin skeleton by injection of an MO-resistant form of E-cadherin mRNA into the cell injected with E-cadherin MO. The asterisk marks the treated cells; #, adjacent untreated cells. Four images are shown of the same field of view. The RLDX (blue) and anti-HA (red) staining show the expression of the HA-tagged mRNA in the same cells as those injected with the MO (co-injected with RLDX), whereas the Phalloidin staining (green) shows increased assembly of F-actin in the injected cells (compare with C, where actin staining is reduced in the MO-injected cells). Scale bars: 100 μm in B,C,G; 20 μm in D,E.
	Fig. 7. Depletion of E-cadherin in the non-neural ectoderm leads to increased thickness and number of cell layers in the epidermis, and to reduction of phosphorlyated myosin light chain in the non-neural ectoderm cells. (A-C) Transverse sections of control and E-cadherin-depleted epidermis at st. 19. The epidermis is thicker and contains increased numbers of cell layers after E-cadherin depletion. Red, MO-injected cells; green, F-actin; EN, endoderm; ME, mesoderm; EC, ectoderm. Thickness is quantitated in C. ***Statistically significant difference in thickness of ectoderm between control and E-cadherin-depleted ectoderm (P<0.001). (D,E) En face views of the non-neural ectoderm in control (D) Xenopus embryos, and in embryos with a large clone of E-cadherin-depleted cells (E). Green, P-MLC; red, E-cadherin. The cells that lack E-cadherin also have reduced levels of P-MLC. Asterisk indicates MO-containing cells. (F,G) Transverse sections through embryos from the same experiment. P-MLC is distributed on all surfaces of the non-neural ectoderm cells in controls (F), but only remains apically in E-cadherin-depleted cells (G). White arrowheads indicate P-MLC on lateral cell membranes; arrows indicate apical P-MLC. Scale bars: 50 μm in A,B,F,G; 100μ m in D,E.
	Fig. 8. N- and E-cadherin cannot replace each other in the neural and non-neural ectoderm. (A,B) The same field of view in brightfield and fluorescence. Injection of N-cadherin MO + RLDX into both dorsal animal cells at the 8-cell stage, thus depleting N-cadherin in most or all of the neural plate, blocks neural fold closure (compare upper row of untreated embryos with middle row of MO-injected embryos). This is rescued by subsequent injection of X. tropicalis (MO-resistant) N-cadherin mRNA into the same cells (lower row of embryos). (C,D) Equivalent pictures to show that injection of E-cadherin mRNA does not rescue neural fold closure. The lower row of embryos has received MO + E-cadherin mRNA. (E) A higher magnification field of view to show that F-actin is not rescued by E-cadherin mRNA (compare control to N-cad MO + E-cad mRNA panels), although the E-cadherin protein is expressed (stained red in the cross-section view with anti-E-cadherin antibody). (F) Four images of the same en face field of view of a clone of cells (asterisk, blue in right-hand panels) depleted of E-cadherin, and subsequently injected with Myc-tagged N-cadherin mRNA. The N-cadherin protein is expressed (stained red with anti-Myc antibody), but does not rescue the loss of F-actin (green) caused by E-cadherin depletion. The cells along the border between untreated cells (#; which express E-cadherin) and cells in which E-cadherin has been replaced by N-cadherin (arrows), do not express N-cadherin on the cell surface adjacent to E-cadherin-expressing cells. Scale bars: 100 μm.
	Fig. 9. N- and E-cadherin may not rescue each other because they localize to different regions of the ectodermal cells. (A) Levels of cortical F-actin in the neural ectoderm in controls, N-cadherin-depleted and N-cadherin-depleted followed by three different doses of E-cadherin mRNA. Cortical actin is not rescued by any dose of E-cadherin mRNA. (B) The converse experiment compares the cortical actin levels in non-neural ectoderm of control, E-cadherin-depleted and N-cadherin depleted followed by three doses of N-cadherin mRNA. Cortical actin is not rescued by any dose of N-cadherin mRNA. (C,D) The levels of E-cadherin and N-cadherin proteins in Xenopus embryos from the same experiment. In each case, translation is efficient and generates at least as much protein as found in the control tissues. ***Statistically significant difference between the control (blue) and labeled bar (P<0.001). (E,F) The location of E-cadherin expressed in N-cadherin-depleted neural ectoderm (E), and N-cadherin expressed in E-cadherin-depleted non-neural ectoderm (F). E-cadherin is expressed basolaterally in the neural ectoderm, whereas N-cadherin is concentrated apically in the non-neural ectoderm. Scale bars: 20 μm.
	Fig. S1. Apical cell surface staining of N-cadherin is specific. (A) Dorsal plate cross-section of a stage 14 embryo injected with 100 pg of Xt-N-cad-Myc mRNA into each dorsal animal cell at the 8-cell stage and subsequently stained for the Myc tag. (B) N-cad immunostaining of a stage 14 neural plate depleted of N-cadherin only in one side by injecting 20 ng of the N-cad MO into a single dorsal animal cell at the 8-cell stage. (C,D) En face and cross section views of stage 14 neural plates stained for β-catenin.
	Fig. S3. Control for N-cadherin staining of E-cadherin MO only injected ventral ectoderms. (A) En face view. (B) Cross-section view.

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