Fig. 1

Lack of Pomca reflects interrenal insufficiency. (a) Schematics of the Pomca knockout (pomc-/-) showing the 14 bp deletion (red letters) in the exon 3 of the Pomca gene. The white box on either end represents the 5??3? upstream and downstream sequences, respectively, and the introns are represented as black lines and exons as a black box; (b) Anatomical view of wildtype, and pomc-/- larvae at 6 days post-fertilization (dpf) showing where the histological sections were obtained for identification of the hypothalamus and pituitary regions. The top panel shows the cross-section of brains stained with hematoxylin and eosin (I and II) and the bottom panels are immunostained for Acth (III and IV) and a-Msh (V and VI); Tectum (Tt), tegmentum (Tg) and hypothalamus (Hp). White arrows indicate Acth- and a-Msh-positive cells. Asterisk (*) denotes the cranial cavity. The rectangle in the insert indicates the hypothalamic and pituitary regions that were magnified. I?II (bar 20 ?m), III?VI (bar 10 ?m) and Insert (bars 40 ?m). The Acth and a-Msh immunoreactivity is absent in the pomc-/- larvae; (c) Representative image of 6 dpf larva showing reduced pigmentation in the pomc-/- larvae (arrowhead) compared to WT (left panel); (d) Quantification of larval pigmentation between the genotypes at 6 dpf (t test, n = 20 larvae per group, ***p < 0.001); (e)The acute cortisol response to a physical stressor (1 min at 250 rpm) was inhibited in the 4 dpf pomc-/- larvae (symbols with different letters are significantly different (2-way ANOVA; n = 8?12, each a pool of 12 larvae); (f) bars represent significant differences in the transcript abundance of mc2r, star, p450ssc, nr3c1, nr3c2, and hsd11b2 in WT and pomc-/- larvae at 6 dpf (t test, n = 6?7, each a pool of 12 larvae, *p < 0.05, **p < 0.01, ***p < 0.001); (g) Anatomical view of wildtype and pomc-/- larvae at 6 dpf showing glucocorticoid receptor (Gr) and mineralocorticoid receptor (Mr) immunoreactivity in the control (i and iii and ii and iv) and cortisol (v and vii and vi and viii) treated groups in the hypothalamus. The panel on the left side shows the anatomy and cross-section of the body stained with Hematoxylin and Eosin (H&E) and the area inside the box indicate where the zoomed images of the brain were acquired to evaluate the immunostaining for Gr and Mr expression (white arrows and *); (h) Cross-section of WT (i, iii, v and vii) and pomc-/- (ii, iv, vi and viii) larvae liver at 6 dpf showing Gr and Mr immunoreactivity with and without cortisol treatment. The panel on the left side shows the anatomy and cross-section of the body stained with H&E to indicate where the zoomed images of the liver were acquired to evaluate the immunostaining for Gr and Mr expression; white arrows indicate Gr- and Mr-positive hepatocytes; spinal cord (SC), notochord (N), muscles (M), head kidney (HK), anterior intestine (I), yolk sac (YS) and liver (L). Bars: 10 µm (i?viii) and 50 µm for the trunk stained with H&E. The transcript abundance and cortisol levels are from whole larvae.