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Fig. S14 Time course of lhx9 and hcrt expression after heat shock. Double fluorescent ISH for lhx9 and hcrt are shown at 1 hour, 8 hours, and 24 hours after heat shock (HS)-induced lhx9 overexpression. Fluorescent ISH for lhx9 and immunostained hcrt:EGFP is shown at 96 hours post HS. (B) At 1 hour post HS, widespread lhx9 mRNA is detected throughout the embryo. Nearly all lhx9-expressing cells also express hcrt. (D) At 8 hours post HS, the number of cells with lhx9 overexpression and ectopic hcrt expression is reduced by approximately four-fold. Most lhx9-expressing cells still express hcrt. (F) At 24 hours post HS, little ectopic lhx9 or hcrt expression is observed, except for ectopic lhx9- and hcrt-co-expressing neurons in the medial hindbrain. Ectopic hcrt-expressing neurons that persist until 96 hours post HS (120 hpf) also express ectopic lhx9 (H, J). In contrast, control embryos injected with empty heat shock vector only exhibit hcrt expression in the hypothalamus (A, C, E, G, I). Arrowheads indicate examples of ectopic lhx9 and hcrt co-expression. (G, H) show 91 µm thick confocal maximum intensity projections containing both endogenous and ectopic Hcrt neurons. (I, J) show 45 µm thick confocal maximum intensity projections including only the region containing ectopic Hcrt neurons. lhx9-expressing neurons that appear close to ectopic Hcrt neurons in (H) are located 30 µm ventral to the ectopic Hcrt neurons, and are thus not observed in (J). Embryos fixed at 1 hour and 8 hours post HS are shown in side view. Embryos fixed at 24 hours and 96 hours post HS are shown dorsally and ventrally, respectively. Boxed regions in (G, H) are shown at higher magnification in (I, J). Scale indicates 50 µm (A-F) and 10 µm (G-J).