Fig 2: Plagl1 expression in embryonic murine brains and localization of PLAGL1 in hypocretin neurons of the embryonic murine hypothalamus. The Plagl1 RNA probe detected Plagl1 expression in periventricular zones and the hypothalamus at E10-E13 by in situ hybridization (red arrowheads) (magnification, x20). Murine HCRT is visualized by staining with Alexa Fluor®488 (green) and murine PLAGL1 is visualized by staining with Alexa Fluor® 594 (red) at E14 and E18. 3vt, third ventricle; E, embryonic day; fv, forebrain vesicle; HCRT, hypocretin; mv, mesencephalic vesicle; PLAGL1, pleomorphic adenoma gene-like 1; tv, telencephalic vesicle.

Fig 3: Relative expression levels of hypothalamic genes following in utero electroporation. In utero electroporation was performed with pCAGGS-EGFP and pCAGGS-mPlagl1 (PLAGL1 group, n=6) to evaluate the effect of PLAGL1 on Hcrt transcription. Control mice were electroporated with pCAGGS-EGFP and pCAGGS (mock group, n=4). Animals were harvested at P0 stage. Following total RNA extraction from the hypothalamus and cDNA synthesis, quantitative polymerase chain reaction was performed. The relative expression levels of each gene were normalized to the relative expression levels of Hprt1. Alterations in transcription levels between the electroporated and contra sides in each group were compared using the Mann-Whitney U-test. Data are presented as the means ± standard deviation. Agrp, agouti related neuropeptide; B2m, ß-2-microglobulin; Contra, contralateral side (no electroporation); EGFP, enhanced green fluorescent protein; EP, electroporated side; Hcrt, hypocretin; Hprt1, hypoxanthine phosphoribosyltransferase 1; Nptx2, neuronal pentaxin 2; Npy, neuropeptide Y; Oxt, oxytocin; Pdyn, prodynorphin; Pmch, pro-melanin-concentrating hormone; Trh, thyrotropin-releasing hormone.

Fig 4: Localization of PLAGL1 in murine hypocretin neurons under several conditions. (A-G) Murine HCRT was visualized by staining with Alexa Fluor® 488 (green) and murine PLAGL1 was visualized by staining with Alexa Fluor® 594 (red). (A) Murine hypothalamic sections at ZT6. The lateral hypothalamic area under (B) ZT6, (C) ZT18 and (D) SD6h conditions, with a relatively specific and uneven distribution of PLAGL1 (arrows). The dorsomedial hypothalamic nucleus under (E) ZT6, (F) ZT18 and (G) SD6h conditions. (H) DAPI-labeled nuclei at ZT6. (I) Alexa Fluor®488-labeled murine HCRT at ZT6. (J) Alexa Fluor® 594-labeled murine PLAGL1 at ZT6. (K) Merged image of (H, blue), (I, green) and (J, red) immunofluorescence at ZT6. (L) DAPI-labeled nuclei at SD6h. (M) Alexa Fluor®488-labeled murine HCRT at SD6h. (N) Alexa Fluor®594-labeled murine PLAGL1 at SD6h. (O) Merged image of (L, blue), (M, green), and (N, red) immunofluorescence at SD6h. Scale bar, (A) 500 µm, (B-G) 40 µm, (O) 10 µm. (H-O) These images are displayed at the same magnification. 3vt, third ventricle; Amy, amygdala; ARC, arcuate nucleus; DMH, dorsomedial hypothalamic nucleus; HCRT, hypocretin; LH, lateral hypothalamus; PLAGL1, pleomorphic adenoma gene-like 1; SD6h, 6 h of sleep deprivation; VMH, ventromedial nucleus of the hypothalamus; ZT, Zeitgeber time.

Fig 5: Double immunohistochemical staining and RNAscope in the murine lateral hypothalamic area at ZT6. (A) PLAGL1 was visualized by Cy5 (white), (B) Plagl1 was visualized by Fast Red (red), (C) HCRT was visualized by Alexa Fluor® 488 (green), (D) and DAPI labeled the nuclei (blue). (E) Merged image of A-D; (F) expanded views of the square field in E. Scale bar, 50 µm. ZT, Zeitgeber time; HCRT, hypocretin; PLAGL1, pleomorphic adenoma gene-like 1.