Fig. 2: Precocious hypothalamic development following elevated GC.
From: Elevated glucocorticoid alters the developmental dynamics of hypothalamic neurogenesis in zebrafish
a Whole-brain IHC for proliferation marker pH3 as seen in a maximum intensity projection in 5 dpf wild-type and star:bPAC+ larvae. Scale bar is 60 μm. b Cell counting of pH3 across brain regions revealed an increase in the hypothalamus (H) of 5 dpf star:bPAC+ larvae, compared with wild type. Wilcox Exact test with Bonferroni correction (N = 14, p = 0.0013195). Brain regions are labelled according to the ZFIN anatomy database. The box shows the median next to the first and third quartile, whilst the whiskers indicate the first and third quartile ±1.5 interquartile range. c Co-expression of rx3, Pcna and pH3 in the hypothalamus of 5 dpf wild-type and star:bPAC+ larvae, observed in a single plane image of FISH-IHC. Scale bar 40 μm. Dashed line indicates the ventricles and arrowheads indicate excess rx3 + pH3+ cells in star:bPAC+ hypothalamus. d IHC for Blbp in the arcuate nucleus of 5 dpf wild-type and star:bPAC+ larvae, observed in a single plane image. Scale bar 40 μm. e IHC for Otpa in the hypothalamus of 5 dpf wild-type and star:bPAC+ larvae, as observed in a maximum intensity projection. Scale bar 40 μm. The hypothalamus of 5 dpf star:bPAC+ larvae is larger (f, t = −2.6193, df = 11.207, p = 0.02353) and has increased intensity of rx3 (g, N = 7, t = −2.3011, df = 8.5909, p = 0.04823) and Pcna (h, N = 7, t = −2.4409, df = 11.766, p = 0.03147). i Excess proliferating pH3+ cells in the hypothalamus of 5 dpf star:bPAC+ larvae are rx3+ radial glia (N = 7, Two-way ANOVA with Tukey’s test: total pH3+ cells (p = 0.0293550), rx3 + pH3+ cells (p = 0.0373125)). Intensity of Blbp (j, N = 9, t = −2.2734, df = 14.903, p = 0.03823) and Otpa (k, N = 12, t = −2.44, df = 16.227, p = 0.02652) was increased in the hypothalamus of 5 dpf star:bPAC+ larvae. l The zebrafish rx3 gene contains two GREs, one in exon one and another in intron one. The region shown is ENSDARG00000052893, GRCz11:21:10755554:10759823:1 (including flanking sequence). m ChIP qPCR analysis of two GREs in the rx3 gene shows enrichment for GR compared with IgG control in 5 dpf wild-type brains. Enrichment of GR was higher in wild types exposed to dexamethasone, and higher still in star:bPAC+ brains for both GREs.
