Fig. 5. Impaired neuronal circuit formation in POMC neurons with defective cilia.

a Axonal projections of tdTomato-labeled POMC neurons in hypothalamic paraventricular nucleus (PVH), dorsomedial nucleus (DMH) and lateral hypothalamic area (LH) of POMC-cre;;KIF3A^f/f;; tdTomato and POMC-cre;;tdTomato male mice at 8 weeks (n = 5). Representative images and the graph depicting the axonal fiber density of POMC neurons in each hypothalamic area. Scale bars: 100 μm. b, c β-Endorphin-immunoreactive axonal projections of POMC neurons in the PVH and DMH of POMC-cre;;IFT88^f/f and POMC-cre/ERT2;;IFT88^f/f mice and their control littermates at 8–10 weeks (n = 6). Scale bars: 100 μm. d The primary neuronal process of tdTomato⁺ POMC neurons in POMC-cre;;KIF3A^f/f;;tdTomato and POMC-cre;;tdTomato mice (n = 6). Representative images and graph depicting the average number of the primary process of 100 POMC neurons. Scale bars: 20 μm. ARH hypothalamic arcuate nucleus. e MAP2 (dendrite marker), neurofilament (axon marker), and tdTomato (POMC neuron) triple immunostaining in primary cultured hypothalamic neurons at 21 days in vitro (DIV), which were obtained from POMC-cre;;IFT88^f/f;;tdTomato and POMC-cre;;tdTomato embryos, respectively. Sholl analysis was conducted for the assessment of dendritic arborization in 20 POMC neurons from POMC-cre;;tdTomato embryos and 12 neurons from POMC-cre;;IFT88^f/f;;tdTomato embryos. The full lengths of axons were measured for 19 cells from POMC-cre;;tdTomato embryos and 8 cells from POMC-cre;;IFT88^f/f;;tdTomato embryos. Scale bars: 20 μm. f NPY immunostaining in the ARH, PVH, and DMH of 3-week-old POMC-cre;;IFT88^f/f mice and IFT88^f/f mice (n = 6 for IFT88^f/f mice, n = 5 for POMC-cre;;IFT88^f/f mice). Scale bars: 100 μm. Data values are presented as mean ± SEM. Statistics were performed using two-sided Student’s t test (a–d, e—axon, f) and one-sided two-way ANOVA followed by post hoc LSD test (e—dendrite). **p < 0.01, ***p < 0.001 between the indicated groups.