Fig. 4: Failed hypothalamic maturation following elevated GC.

a Expression of rx3, Pcna and Blbp in the hypothalamus of 13 dpf wild types and star:bPAC larvae, observed via FISH-IHC in single plane confocal images through the hypothalamus. Scale bar is 40 μm. b-e Quantitative analysis of confocal images shown in (a), N = 8. Volume of the hypothalamus is not different (b, t = 0.31771, df = 10.899, p = 0.7567), meanwhile, rx3 CTCF shows a trend towards reduction (c, t = 1.8613, df = 8.3108, p = 0.09835), Pcna CTCF is significantly reduced (d, t = 4.0207, df = 7.7651, p = 0.00408), and Blbp CTCF is not significantly different (e, t = −1.2827, df = 13.092, p = 0.2219) in the hypothalamus of 13 dpf star:bPAC+ larvae compared with wild types. f-g Quantitative analysis of co-expression of Pcna in hypothalamic radial glia, from the images shown in (a). Blbp cells (f, t = −2.5888, df = 7.5126, p = 0.0339) and rx3 cells (g, t = 4.7867, df = 13.444, p = 0.0003238) have a significant reduction in Pcna co-expression in the hypothalamus of 13 dpf star:bPAC+ larvae compared with wild types. h Expression of genes involved in feeding regulation in the hypothalamus of 13 dpf star:bPAC+ and wild-type larvae as shown in maximum intensity projections of confocal stacks. Scale bar is 40 μm. i−k Quantitative analysis of cell counts in the stacks shown in (h). Number of agrp cells is significantly lower (i, N = 9 wild type, N = 6 bPAC + , t = 2.2891, df = 11.255, p = 0.04234), number of cart4 cells is significantly reduced (j, N = 20 wild type, N = 21 bPAC+ across 2 independent experiments, t = 2.8544, df = 34.862, p = 0.007212), number of hcrt cells shows a trend reduction (k, N = 15 wild type, N = 18 bPAC+ across 2 independent experiments, t = 1.9553, df = 26.571, p = 0.06115). All graphs show the mean with standard error. #p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001.