Fig 2: Loss of CDH11 reduces definitive NC cells. To determine if loss of CDH11 affected neural progenitors and definitive NC cells in addition to NC progenitors, embryos were injected with CDH11MO or ContMO and electroporated, and IHC was performed for definitive NC cells (SOX9, SNAI2, SOX10) and neural progenitors (SOX2). (A) IHC for SOX9 in transverse section from HH8 embryo with (B) overlay with CDH11MO (green). (C) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of SOX9+ cells is 20.34 on uninjected and 11.85 on CDH11MO-injected side, p = 0.04, n = 11. (D) IHC for SNAI2 in transverse section from HH9 embryo with (E) overlay with CDH11MO (green). (F) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of SNAI2+ cells is 22.75 on uninjected and 13.75 on CDH11MO-injected side, p = 0.001, n = 16. (G) IHC for SOX10 in transverse section from HH8 embryo with (H) overlay with CDH11MO (green). (I) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of SOX10+ cells is 20.35 on uninjected and 11.87 on CDH11MO-injected side, p = 0.01, n = 18. (J–M’) Overlay from the same embryo to demonstrate reduction in NC progenitors (PAX7) and definitive NC cells (SOX9, SOX10) after CDH11 MO-injection (n = 8/9 embryos with reduced cells). (N) IHC for SOX2 in transverse section from HH8 embryo with (O) overlay with CDH11MO (green). (P) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of SOX2+ cells is 80.20 on uninjected and 79.50 on CDH11MO-injected side, p = 0.90, n = 5. All graphs show mean (indicated on graph) and median (line within graph). Scale bar for (A,B,D,E,G,H,N,O) indicated in (A) and scale bar for (J-M’) indicated in (J). Reducing CDH11 significantly reduces the entire population of premigratory NC cells without affecting the SOX2-positive neural tube progenitors.

Fig 3: MKN45P-PR cells demonstrates metastatic and PTX-resistant characteristics. (A) CDH11 and E-cadherin expression were examined by western blot. (B-D) A matrigel invasion and migration assay and a scratch-wound assay (E, F) were performed. The wound closure (%) was calculated (mean ± SD, *P < 0.05). (G) MKN45P and MKN45P-PR cells were intraperitoneally injected into the nude mice, and treated with vehicle and PTX; macroscopic images of the peritoneal disseminations are shown, and the number of peritoneal nodules from each mouse were measured (H) (mean ± SD, *P < 0.05).

Fig 4: Summary diagram of CDH11-knockdown phenotype. (A) Image depicting NC cell development in normal NC cells (CDH11+) and cells lacking CDH11 (CDH11-). Normal cells undergo EMT, exit the neural tube, migrate collectively, and then progressively mesenchymalize as development proceeds. Normal lamellipodial and filopodial projections form as the cells navigate through the extracellular matrix. In the absence of CDH11, NC cells are induced, but undergo p53-mediated apoptosis due to either (1) inability to complete EMT/migration or (2) altered intracellular signaling in the absence of CDH11. PAX7, SOX9, SNAI2, and SOX10 positive cells are all significantly reduced in the absence of CDH11 while p53 and *Casp3 are upregulated. (B) Simplified NC GRN identifying NC specifiers and multiple putative inputs into the CDH11 upstream regulatory region as identified by ATAC-seq performed on NC cells (Williams et al., 2019). Little is known about the downstream targets and effectors of most cadherin proteins in the NC GRN with the exception of CDH6B (Schiffmacher et al., 2016) and CDH11 in migratory NC cells (Kashef et al., 2009; Koehler et al., 2013), and therefore identifying their targets in premigratory NC cells is essential. Direct binding relationships are indicated with solid lines while putative regulatory relationships are indicated by dashed lines. Letters indicate species in which experiments were performed: x = Xenopus, c = chicken, m = mouse. GRN information sourced from Rogers and Nie (2018).

Fig 5: Exosomes carrying miR-127-3p inhibit CDH11 expression in chondrocytes. (A) Effects of IL-1ß and exosome treatment on the expression of CDH11 in chondrocytes detected by RT-qPCR. (B) RT-qPCR for detecting the transfection efficiency of pcDNA-CDH11. (C) Effects of upregulated CDH11 on the viability of chondrocytes measured by MTT assay. (D) Effects of upregulated CDH11 on the DNA synthesis activity of chondrocytes measured by EdU assay. (E) Effects of upregulated CDH11 on the apoptosis rate of chondrocytes determined by flow cytometry. (F) Effects of upregulated CDH11 on the expression of Collagen II and MMP13 in chondrocytes determined by Western blot assay. All the experiments were performed independently in triplicate and the results were the mean of three values. Unpaired t test (B–E) was utilized for comparison between two groups or one-way ANOVA (A)/two-way ANOVA (F) was used for comparison among multiple groups. *p < 0.05 vs control group; #p < 0.05 vs OA group; &p < 0.05 vs Exo + pcDNA group.