Fig 1: Organisation of tuberal domains through a neuroepithelial-intrinsic mechanism.(A–F) MIPs of hemi-dissected heads after HCR, showing expression of BMP2 or BMP7 at HH10, HH16 and HH17. Expression is detected in preplacodal ectoderm/Rathke’s pouch (blue arrows) and the neuroectoderm (pink arrows). Dots in (A, B, D and E) outline basal neural tube. (G–I) Steps describing hypothalamic tissue isolation from an HH10 embryo: (G, G′) filleting, yellow arrows in (G) mark the position of the slice containing hypothalamic tissue, shown at higher power in (G′). (H–H″) Isolated slices containing hypothalamic tissue (pink) have a characteristic shape, and the prechordal mesoderm can be identified morphologically (dotted outline in H). After dispase treatment, neural tissue is isolated from surrounding tissues (H′), and hypothalamic tissue is excised (H″). (I) Isolated hypothalamic tissue is embedded in a 3-D collagen matrix. (J–N””) Sections through hypothalamic explants, at 0 hr, or cultured for 24 or 96 hr, analysed by HCR or immunolabelling. (J) At 0 hr, pSMAD1/5/8 and SHH are detected throughout the section (single channel views shown in (J′–J′′′), blue shows DAPI counterstain). (K) At 0 hr, FGF10 and SHH are detected throughout the section single channel views shown in (K′, K″). (L–M) After a 24-hr culture period, to the equivalent of HH13, FGF10 and SHH begin to resolve ((L): single channel views shown in (L′, L′′)), and discrete domains of SIX6, SHH and TBX2 are apparent ((M): double channel views shown in (M′, M″)). (N) Brightfield image of explant cultured for 96 hr to the equivalent of HH24. (N’-N””) Same explant, after immunolabelling, shows organised expression of ISL1, SHH, pSMAD1/5/8 and EMX2 ((N′): double channel views shown in (N″–N″″)). (O–P) Serial adjacent sagittal sections of HH19 embryos, analysed by HCR to show expression of SHH, FGF10 and EMX2 ((O, O′) shows same section without FGF10), or by immunolabelling to detect ISL1 and pSMAD1/5/8 (P). (Q) Schematic summarising the expression of SHH, TBX2, FGF10, ISL1, EMX2, pSMAD1/5/8 and BMP2/7 at HH19. Scale bars: 100 μm. Each panel shows a representative image from a minimum of n=3 embryos or explants. HCR, hybridisation chain reaction; MIP, maximum intensity projection.
Fig 2: BMP promotes tuberal identity in HypFP cells.(A, D) HH6 (A) or HH8 (D) isolated neuroepithelia, after HCR to detect NKX2-1 and SHH. Boxes show regions dissected for explant culture. (B–B′′′′′′, C–C′′′′′′′, E–E′′′′′, F–F′′′′′) HH6 anterior or posterior explants cultured to a HH14 equivalent in control media (rows B, C, E) or 32 nM BMP2/7 protein (row F), and then processed through repeated rounds of multiplex HCR for SIX6, SHH, TBX2/NKX2-1/NR5A1/POMC or SIX6/FOXA2/SHH/TBX2/NR5A1. (G–H) HH8 posterior explants cultured to an HH18 equivalent in control media (G) or BMP2/7 (H) and processed by wholemount HCR for POMC. Scale bars: 100 µm. NKX2-1, NR5A1, POMC and FOXA2 were analysed in a minimum of n = 3 explants/condition; SIX6, SHH and TBX2 were analysed in a minimum of n = 11 HH6 explants/condition and n = 7 HH8 explants/condition. HCR, hybridisation chain reaction.
Fig 3: Ectopic BMP exposure reduces anterior, then posterior tuberal progenitors.(A–D) MIPs of hemi-dissected head of HH13/14 embryo taken through two rounds of multiplex HCR analysis, after implantation of PBS bead (A) (single channel views in A′–A″′, B) or BMP2/7-soaked bead (C) (single channel views in C′–C″′, D). In (A–C″′), white bracket shows the anterior tuberal domain, absent after BMP2/7 exposure; pink bracket shows the posterior tuberal domain; yellow arrows show anterior ID. In (B, D), white arrows point to FST+ve cells, absent after exposure to BMP2/7. (E) Graph showing a significant increase in intensity of TBX2 following BMP2/7 bead implantation p < 0.05, unpaired t test; error bars show SEM; n = 3 embryos/condition. (F–J′; L–P′; R–V′) Hemi-dissected heads of HH19 embryos after implantation of PBS bead (F–V) or BMP2/7-soaked bead (F′–V′). Each panel shows MIP of single channel views (multiplex views shown in Figure 6—figure supplement 1). In (F, F′), yellow arrows indicate the length of the SIX6+ve domain. Dotted outlines in (G, G′) show area of SHH expression, measured in (K). Yellow dotted outlines in(N, N′, T, T’, U, U’, V, V’) show area of anterior and tuberal hypothalamus, measured in (Q). White dotted outlines in (O, O’ P, P’ and V, V’ ) show area of optic stalk entrance, measured in ( W). (K, Q) There is a significant decrease in the area of SHH expression, p < 0.0001****, unpaired t test and a significant decrease in the area of anterior and posterior tuberal hypothalamus, p < 0.0004***, unpaired t test between PBS and BMP2/7 bead-implanted embryos. n = 6 embryos/condition; error bars show SEM. (W) There is a significant increase in the area of optic stalk entrance, p < 0.001**, unpaired t test. n = 4 embryos/condition; error bars show SEM.. (X–Y′) Serial adjacent sagittal sections of HH17 embryos, immunolabelled to detect expression of SHH, pSMAD1/5/8 or the M-phase marker phosphoH3 (pH3) after implanting PBS bead (X, X′) or BMP2/7-soaked bead (Y, Y′) at HH10. (Z) Graph showing significant decrease in the number of pH3 positive M-phase progenitors (p < 0.05*, unpaired t test; n = 5 embryos/condition; error bars show SD). Scale bars: 100 µm. HCR, hybridisation chain reaction; MIP, maximum intensity projection. Figure 6—source data 1.Raw data for Figure 6E. Figure 6—source data 2.Raw data for Figure 6K, Q and W. Figure 6—source data 3.Raw data for Figure 6Z.
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