Fig 2: 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 μm), III–VI (bar 10 μm) and Insert (bars 40 μm). 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.

Fig 3: Effect of siMEG3 on the bioactivity of PitNET cell lines. (A) RT-qPCR experiment measured the RNAi efficiency of siMEG3-1 and siMEG3-2. (B) Western blot experiment showed RNAi-MEG3 reduced the level of DLK1, PIT1 and GH in GH3 cell line. (C) RNAi-MEG3 obviously increased the cell viability of GH3 cell line, mildly increase in MMQ cell line, and no change in ATT20 cell line. (D) RNAi-MEG3 inhibited the secretion of GH/IGF-1 in GH3 cells, not PRL in MMQ cells and ACTH in ATT20 cells. (E) Confocal experiment showed RNAi-MEG3 inhibited the levels of DLK1 and PIT1 in GH3 cells. *compare to control group P<0.05 **P<0.01 ***P<0.001.

Fig 4: Effect of subcutaneous injection of glucocorticoid on the hypothalamus-pituitary-adrenal gland axis in 4-week-old chickens. The expression of NPBWR2 (A) and POMC (B) in the pituitary gland and NPW (C) in the hypothalamus after subcutaneous injection of dexamethasone [0.4 mg/100 g body weight (BW)/day, 0.5 mL for short-term evaluation (3 hours); 0.2 mg/100 g BW/day, 0.5 mL for long-term evaluation (7 days)]. The concentrations of adrenocorticotropin (D) and corticosterone (E) in plasma after subcutaneous injection of dexamethasone. Each data point represents the mean ± SE of the mean of 6 replicates. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control group.

Fig 5: Immunofluorescence staining for detecting autoantibody. Immunofluorescence staining of patient serum and anti-ACTH antibody in the mouse pituitary. (A) Serum from the patient with specifically recognized corticotrophs compared with sera from healthy subjects. Pre-absorption of the patient’s serum with recombinant human POMC protein diminished reactivity to corticotrophs, indicating the presence of circulating anti-POMC antibodies in the serum. (B) Pre-absorption of POMC or ACTH1-39 protein into the serum diminished the signal, whereas pre-absorption of ACTH1-24 or α-MSH protein showed no signal reduction, indicating that ACTH25-39 was the region recognized by the circulating anti-POMC antibody. The representative results are presented in the figure. Scale bar 50 µm. ACTH, adrenocorticotropic hormone; POMC, proopiomelanocortin; α-MSH, alpha-melanocyte-stimulating hormone.