Nectin-2 and N-cadherin interact through extracellular domains and induce apical accumulation of F-actin in apical constriction of Xenopus neural tube morphogenesis

Abstract

Neural tube formation is one of the most dynamic morphogenetic processes of vertebrate development. However, the molecules regulating its initiation are mostly unknown. Here, we demonstrated that nectin-2, an immunoglobulin-like cell adhesion molecule, is involved in the neurulation of Xenopus embryos in cooperation with N-cadherin. First, we found that, at the beginning of neurulation, nectin-2 was strongly expressed in the superficial cells of neuroepithelium. The knockdown of nectin-2 impaired neural fold formation by attenuating F-actin accumulation and apical constriction, a cell-shape change that is required for neural tube folding. Conversely, the overexpression of nectin-2 in non-neural ectoderm induced ectopic apical constrictions with accumulated F-actin. However, experiments with domain-deleted nectin-2 revealed that the intracellular afadin-binding motif, which links nectin-2 and F-actin, was not required for the generation of the ectopic apical constriction. Furthermore, we found that nectin-2 physically interacts with N-cadherin through extracellular domains, and they cooperatively enhanced apical constriction by driving the accumulation of F-actin at the apical cell surface. Interestingly, the accumulation of N-cadherin at the apical surface of neuroepithelium was dependent on the presence of nectin-2, but that of nectin-2 was not affected by depletion of N-cadherin. We propose a novel mechanism of neural tube morphogenesis regulated by the two types of cell adhesion molecules.

Fig. 1.

Expression pattern of nectin-2 during neurulation. (A-F) Expression pattern of nectin-2α in stages of early neural fold (stage 15; A,B), mid-neural fold (stage 16; C,D) and late neural fold (stage 18; E,F). (G,H) Negative control with sense probe (stage 15). Dorsal surface (A,C,E,G) and transverse sections through the trunk region (B,D,F,H) were observed. Arrowheads: nectin-2α expression in the superficial layer of the neuroepithelium. Dotted lines: borders between ectoderm (outer layer), notochord and prospective somites. no, notochord; so, somites.

Fig. 2.

Knockdown of nectin-2 disrupts neural fold formation. (A) Nucleotide sequences of nectin-2 constructs and nec2-MO. Asterisks: nucleotides identical in each construct and nec2-MO. (B) Depletion of nectin-2 by nec2-MO. Lysates from injected embryos were subjected to western blot. (C) Resistance of nec2-res to nec2-MO. Lysates from injected embryos were subjected to western blot. (D-I) Phenotypes of developing neural fold in uninjected (D,E), nec2-MO-injected (F,G) or partially rescued (H,I) embryos. Asterisks: injected side. Arrowheads: neural ridges. (J) Summary of phenotypes. The MOs were injected at 0.25 pmol (B,C,F-J).

Fig. 3.

Apical constriction of neuroepithelium is inhibited by nec2-MO. (A,A′) Section through an embryo given a unilateral injection of nec2-MO. Arrow: cells on the uninjected side undergoing apical constriction. Asterisk: nec2-MO-injected side. Tubulin staining was used to trace the cortices of superficial cells (A′). (B,C) Ratio of apical surface length to perimeter (B) and cell height along the apicobasal axis (C). Each datum was obtained from 15 cells of three embryos. Nec2-res mRNA was injected at 12.5 pg. Error bars: mean ± s.e.m. (D-G) Dorsal surface views of the trunk region in control MO (D,E) and nec2-MO (F,G) injected embryos stained by phalloidin. White brackets: MO-injected side. (H-K) Sections of control MO (H,I) and nec2-MO-injected (J,K) embryos stained by phalloidin. Arrows: MO-containing superficial cells. MOs were injected at 0.25 pmol. Scale bars: 20 μm in A,H,J; 50 μm in D,F.

Fig. 4.

Overexpression of nectin-2 induces ectopic apical constriction. (A-D) Surface views of non-neural ectoderm of control (A,B) and nectin-2 mRNA-injected (C,D) embryos at stages 13-14 (early neurulae). B and D are magnified views of white boxes in A and C, respectively. (E-H) Non-neural ectoderm of control (E,F) and nectin-2-injected (G,H) embryos. Arrows: apically constricted cells. (I-L) Sections showing non-neural ectoderm of control (I,J) and nectin-2-injected (K,L) embryos. Arrows: apically constricted cells. (M) Aberrant surface phenotype in control (memGFP-injected) and nectin-2-injected embryos. Error bars: mean ± s.e.m. (N,O) Ratio of apical surface to perimeter (N) and cell height along the apicobasal axis (O). Error bars: mean ± s.e.m. (P) F-actin staining of normal neuroepithelium at stage 16 (mid-neurula). (Q,R) Subcellular localization of nec2-FLAG in non-neural ectoderm. Arrows: apically localized nectin-2. 100 pg of each mRNA was used, except for nectin-2 in M (100 pg or 200 pg) and nec2-FLAG in Q,R (20 pg). Scale bars: 50 μm.

Fig. 5.

The extracellular domain of nectin-2 is required for apical constriction. (A) Schematic drawing of nectin-2 deletion constructs. (B) Aberrant surface phenotype in uninjected control and nec2-ΔC4-, -ΔIC- or -ΔEC-injected embryos. Error bars: mean ± s.e.m. (C-H) Surface views of non-neural ectoderm of embryos injected with nec2-ΔC4 (C,D), nec2-ΔIC (E,F) or nec2-ΔEC (G,H), observed at stages 13-14 (early neurulae). D, F and H are magnified views of the areas enclosed by white boxes in C, E and G, respectively. (I-N) Cell shape and F-actin staining of embryos injected with nec2-ΔC4 (I,J), -ΔIC (K,L), -ΔEC (M,N). Arrows: apically constricted cells. (O-T) Sections through embryos injected with nec2-ΔC4 (O,P), -ΔIC (Q,R) or -ΔEC (S,T). Arrows: apically constricted cells. (U) Rescue experiments for nectin-2 depletion with the nectin-2-deletion constructs. mRNAs were injected at 12.5 pg, and nec2-MO was 0.25 pmol. Error bars: mean ± s.e.m. ^*P<0.05, n.s.: not significant, t-test. 100 pg of each mRNA was injected (B-T). Scale bars: 50 μm.

Fig. 6.

Nectin-2 interacts with N-cadherin, enhancing apical constriction. (A) A GST pull-down assay with nec2-ex and Ncad-ex, Ecad-ex or Ccad-ex performed using the 293T cell line. (B-G) Overexpression of nectin-2 or N-cadherin in non-neural ectoderm. Low doses of nectin-2 (50 pg; B,C) or N-cadherin (50 pg; D,E) alone and in combination (F,G) were injected and observed at stages 13-14 (early neurulae). Arrows: apically constricted cells. (H) Embryos with aberrant surface in the overexpression experiments with 50 pg of nectin-2 and 50 pg of N-, E- or C-cadherin. Error bars: mean ± s.e.m. (I) Nectin-2 (50 pg) and N-cadherin (50 pg) were separately injected into adjacent blastomeres of four-cell-stage embryos with memGFP and memRFP, respectively. The injected embryos were observed at the early neurula stage. (J-M) Overexpression of E- (J,K) or C-cadherin (L,M) with or without 50 pg of nectin-2. Arrows: apically constricted cells. Scale bars: 50 μm.

Fig. 7.

N-cadherin is functionally associated with nectin-2 in the apical constriction of the neuroepithelium. (A,B) F-actin staining of Ncad-MO-injected embryos. Dorsal (A) and section (B) views. White brackets: MO-injected side. Arrows: MO-containing superficial cells. Ncad-MO was injected at 1.2 pmol. (C) Embryos with defective neural fold in the knockdown with either nec2-MO or Ncad-MO, or both. Control MO and nec2-MO were injected at 0.13 pmol; Ncad-MO was 0.6 pmol. Error bars: mean ± s.e.m. ^*P<0.05, ^***P<0.001, t-test. (D-G) Localization of N-cadherin in the neuroepithelium of control (D,E) and nec2-MO-injected (F,G) embryos. MOs were injected at 0.25 pmol. (H-K) Localization of nec2-FLAG in the neuroepithelium of control (H,I) and Ncad-MO-injected (J,K) embryos. Nec2-FLAG mRNA was injected at 20 pg, and Ncad-MO was 1.2 pmol. (L) Defective neural fold phenotype in embryos injected with 50 pg of full-length N-cadherin or Ncad-Δβ mRNA. Error bars: mean ± s.e.m. ^*P<0.05, t-test. (M) A rescue experiment for N-cadherin depletion with Ncad-res or Ncad-res-Δβ. mRNAs were injected at 50 pg and Ncad-MO was 1.2 pmol. Error bars: mean ± s.e.m. ^*P<0.05, t-test. (N-Q)F-actin staining of neural ectoderm in control (N,O) and Ncad-Δβ-injected (P,Q) embryos. Ncad-Δβ mRNA was injected at 50 pg. Scale bars: 20 μm, except for 100 μm in A.