Fig. S1

Characterisation of Et(-1.0otpa:mmGFP)hd1 transgenic embryos.

(A-D) Dorsal views, Maximum Intensity Projections (MIPs), anterior to the top, focussed on the habenular nuclei of Et(1.0otpa:mmGFP)hd1 transgenic embryos. GFP expression (green) overlaps with (A) acetylated tubulin, (B) kctd12.1, (C) kctd8 and (D) kisspeptin-1 (red). (E) Lateral view, anterior to the left, MIP. Habenular efferent projections innervating their targets in the ventral midbrain at 4 dpf in an Et(1.0otpa:mmGFP)hd1 embryo. (F) Dorsal view, anterior to the left, MIP. Axonal projections innervating the MR (arrowheads) in an 3 dpf Et(1.0otpa:mmGFP)hd1 embryo. Nuclei (blue) are labelled with H2A-CFP. (G) Dorsal view, anterior to the left, MIP. Et(1.0otpa:mmGFP)hd1 embryo showing the emergence of GFP in dHb cells. Anterior and posterior white arrowheads show the dHb and ThEPC cells respectively. Green arrowheads show early dHb efferent axons extending into the ThEPCs. (H) Dorsal view with anterior to the top, MIP, of a 52 hpf Et(-1.0otpa:mmGFP)hd1 embryo labelled with cxcr4b (red) expressed in dHb progenitor cells. Yellow arrowheads indicate the dHb region containing cxcr4b positive cells and transgenic GFP expressing cells. White arrowheads mark the ThEPCs. (I-L) Lateral views focussed on the thalamic area, MIPs of 32 hpf Et(- 1.0otpa:mmGFP)hd1 embryos co-labelled (red) with (I) the prosomere 2 (dorsal thalamus and epithalamus) marker lhx2b [S1, S2], (J) the calcium binding protein Calretinin expressed in the nucleus of the median longitudinal fascicle [S3] and (K, L) the pretectal markers lhx1a and nr4a2a [S4-S6]. Images were cropped and the gamma was adjusted for display purposes. d, dorsal; dHbl, lateral dorsal habenula; dHbm, medial dorsal habenula; dpf, days post fertilization; HC, habenular commissure; IPN, interpeduncular nucleus; l, left; MR, median raphe; Nmlf, nucleus of the medial longitudinal fascicle; PT, pretectum; r, right; T, telencephalon; Tec, optic tectum; ThEPC, thalamic-epithalamic early projecting cluster; v, ventral; VT, ventral thalamus.