Fig. 2.

Anterior/tuberal hypothalamus elongates from rx3⁺ progenitors. (A-H′) Side views after single or double FISH at 30 hpf (A-D) and 55 hpf (E-H; E′-H′ show high-power views of boxed regions). Arrows in A,C,E,G show distances measured for growth comparisons. Arrowheads in E′,F′ indicate position of recesses (colour-coded as in Fig. 1A). (I-K) Maximum intensity projections of representative sections through 30 hpf embryos. I,J show serial adjacent sections; I′,J′ show single-channel views. Arrowheads show co-labelled (yellow) or single-labelled (green) cells. T-shaped white dotted lines indicate outline of AR and LR. (L,L′) Side views of 55 hpf embryo; L′ shows single-channel view. (M,M′) Representative single-plane views taken through zone II; M′ shows single-channel view. Yellow arrowheads show double-labelled cells; green arrowheads point to phosH3⁺ rx3⁻ cells at recess tips. (N) Quantitative analyses of cycling cells at 30-55 hpf as indicated by phosH3 expression in rx3⁺ cells, rx3⁻ cells or in cells adjacent (adj.) to rx3⁺ cells. (O) Schematic depicting rx3, fgf3 and emx2 expression, and change in length and axial orientation of hypothalamus. A ‘bending’ of the tuberal/anterior hypothalamus occurs over 30-55 hpf, relative to the rostro-caudal axis. Red arrows indicate length of dorsal diencephalon or length of emx2⁺ PH; white arrows indicate length of rx3⁺ territories; blue arrows indicate distance from DTJ to rx3⁺ zone III. (P) Length from DTJ to rostral tip of rx3⁺ zone III (n=5 embryos each at 30, 40, 48, 55 hpf). (Q) Tuberal/anterior hypothalamus grows approx. 2.5-fold more than dorsal diencephalon, emx2⁺ PH or ventral rx3⁺ zone III (n=10 each; P<0.0001). Dotted and dashed lines delineate ventral hypothalamus and T-shaped AR/LR (white), adenohypophysis (blue) and rx3-expressing domain (red). AH, anterior hypothalamus; dA, dorso-anterior; PH, posterior hypothalamus; TH; tuberal hypothalamus; vT, ventral tuberal. Scale bars: 50 µm.