Fig 2: Phox2b-Ntrk2-/- mice are perinatal lethal and Phox2b-Ntrk2+/- are similar in body weight, adiposity and length compared to wild type controls on HFD. (A) Representative image of Phox2b-Ntrk2-/- mice and wild type control littermates at P18. (B) Representative immunofluorescence of Phox2b+ and TrkB+ cells in the NTS of control (top) and Phox2b-Ntrk2-/- (bottom) mice. White arrows indicate representative cells that are Phox2b+ and TrkB+. (C) TrkB protein levels in the NTS-enriched lysates (top 2 blots) and the hypothalamus (bottom 2 blots) of Phox2b-Ntrk2+/- (Het) and Cre-controls (WT). SHP2 protein levels are shown as a loading control. (D) Blots are quantified using ImageJ software, n = 7 for each genotype. (E) Body weights of female Phox2b-Ntrk2+/- (n = 9) mice and wild type controls (n = 17) on HFD. (F) Gonadal fat weight of female Phox2b-Ntrk2+/- (n = 9) mice and wild type controls (n = 11). (G) Body length for female Phox2b-Ntrk2+/- (n = 9) mice and wild type controls (n = 11). All values are mean ± SEM. Weight curves are analyzed by two-way ANOVA followed by Bonferroni post-hoc pairwise comparison between Phox2b-Ntrk2+/- mice and wild type controls. Body composition and body length data are analyzed by unpaired two-tailed Student's t-test. *p < 0.05 compared to wild type.

Fig 3: Netrin1 induces neuronal differentiation through the Hand2/Phox2b signal axis. (A) Knockdown efficiency of Phox2b in mouse neural stem cells (NSCs) evaluated via Western blotting. shRNA3# showed the most effective Phox2b knockdown. (B) Histograms showing statistical analysis for Phox2b expression in the 500 ng/mL Netrin1-treated BMSC and control groups. (C) Expression levels of the neuron markers microtubule-associated protein (Map2), neurofilament (NF), and ß3-tubulin following Phox2b downregulation were measured in NSCs cultured with or without 500 ng/mL rNetrin1. Histograms showing statistical analysis for the relative expression of Map2 (D), NF (E), and ß3-tubulin (F). (*: P < .05, **: P < .01, ***: P < .001, ****: P < .0001).

Fig 4: Mining functional proteins involved in Netrin1-induced neuronal differentiation via GEO and bioinformatic analyses, and verification of Phox2b and Hand2 expression induced by Netrin1 in BMSCs and NSCs, respectively. (A) Heat map of differentially expressed genes (DEGs) related to neural crest cell differentiation. (B) Protein–protein interaction network analysis of upregulated DEGs. (C) Western blots showing the expression levels of Hand2 and Phox2b in the 500 ng/mL rNetrin1-treated NSC group. Histograms showing statistical analysis for the relative expression of Phox2b (D) and Hand2 (E) in the 500 ng/mL rNetrin1-treated NSC and the control groups. (F) Expression levels of Hand2 and Phox2b in the 500 ng/mL rNetrin1-treated BMSC group. Histograms showing statistical analysis for the relative expression of Phox2b (G) and Hand2 (H) in the 500 ng/mL Netrin1-treated BMSC and control groups (*: P < .05; **: P < .01; ***: P < .001) .