Fig 2: Inhibition of hypothalamic induction by prethalamic-like derived follistatin(A and B) Immunohistochemical analysis of PAX6 and NKX2–1 in HH4 cultured explant (A–A”) or HH6 cultured explant (B–B”) (n = 5 explants/condition).(C) Schematic depicts ex vivo explant experiments and in vivo gain- or loss-of-function experiments.(D–F) In situ HCR showing SHH, NKX2–1, and PAX6 in sagittal sections of HH14 embryos after in vivo injection (C, bottom schematic) of PBS (D), anti-FST (E), or FST (F) at HH5–HH6. n = 3 embryos/condition.(G and H) In situ HCR showing SHH, NKX2–1, and PAX6 in sections of explants cultured from HH4–HH14 (C, top schematic) in either control medium (G) or anti-FST (H).(I and J) HCR showing SHH, NKX2–1, and PAX6 in sections of explants cultured from HH6–HH14 (C, middle schematic) in either control medium (I) or anti-Fst (J). n = 7–10 explants/condition.(K) Schematic showing FST regulation of hypothalamus development.Scale bars, 100 μm.

Fig 3: Specification of hypothalamic identity at HH8–HH10(A–C and M–O) UMAP plots showing: HH8 (A) and HH10 (M) clusters and annotations; HH8 (B) and HH10 (N) representative gene expression profiles demarcating hypothalamic floor-plate-like and prethalamic-like clusters; and HH8 (C) and HH10 (O) scRNA-seq trajectories obtained from RNA velocity.(D and P) Schematic diagrams (ventral views) showing prethalamic and hypothalamic regions relative to other progenitor domains at HH8 (D) and HH10 (P), color-coded to match UMAP plots.(E–L and Q–V”) Maximum intensity projections showing ventral views after wholemount in situ HCR on isolated neuroectoderm at HH8 (E–L) or HH10 (Q–V) for combinations of transcription factors and ligands (n = 5–15 each). Embryos shown in (H) and (K) are 5- and 3 somites, respectively: in each, NKX2–1 and FST show similar posterior boundaries along the A-P axis. NKX2–1/FST co-localize (R = 0.41, analysis conducted on the entire field of view shown in K).(W and Y) Transverse sections after in situ HCR to detect NKX2–1/PAX6. High magnification views of boxed regions shown in (W) and (Y) used as regions of interest (ROIs) for co-localization (R = 0.23).Scale bars, 100 μm. Ant, anterior; Dor, dorsal; FP, floor plate; Lat, lateral, Med, medial; NC, neural crest; Post, posterior; Telen, telencephalon. Other abbreviations as per Figure 1.

Fig 4: Western blotting of signaling pathway proteins following WAT browning and blood glucose metabolism in beige adipose.A, a representative western blotting of the AMPK-PGC1α-FNDC5 signaling pathway proteins. B, the expression levels of key proteins in the AMPK-PGC1α-FNDC5 pathway were significantly higher in the FSTW group compared with the PBSW group. C, the level of phosphorylation of AMPK. D, western blotting of ERK1/2 and phosphorylated ERK1/2. E, the phosphorylation level of ERK1/2 was significantly higher in the FSTW group compared with the PBSW group. F, western blotting of insulin signaling pathway proteins. G, the expression levels of IR, AKT, P-AKT and GLUT4 were significantly increased in the FSTW group compared with the PBSW group. H, western blotting of the proteins associated with mitochondrial activity. I, the expression levels of UCP1, ATP5A, UQCRC2, SDHB were significantly higher in the FSTW group compared with the PBSW group. Bars represent the mean ± SEM, n = 6. *p < 0.05 and **p < 0.01 when the FST injection group was compared with the controls. Abbreviations: PBSW (PBS withdrawal), the group received PBS injection for one week and no PBS injection for the next week. FST, the group received one week of follistatin injection and was then sacrificed in the FST withdrawal experiment (independent of the FST group in the FST injection experiment). FSTW (follistatin withdrawal), the group received one week of FST injection and no injections for the next week.

Fig 5: Effect of FST injection on body weight and blood glucose metabolism in DIO mice.A, FST injected mice lost weight after one week compared to the control group. B, FST injection decreased body fat index. C, No significant difference in food intake was observed between the FST injection group and the control group. D, GTT, FST injection increased the level of blood glucose metabolism in DIO mice. E, area under the curve of GTT. F, HOMA-IR, FST injection reduced insulin resistance in DIO mice. G-H, rectal temperature and infrared imagery of mice in the 4°C environment: FST injection group maintained a higher body temperature in the cold environment compared to the control group. Abbreviations: FST, the FST injection group, which received one-week injection of FST. Control, the control group, which had no treatment in the FST injection experiment. Bars represent the mean ± SEM, n = 6. *P < 0.05 and **P < 0.01 when the FST injection group was compared with the controls.