Fig 2: 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.