Fig. S3

Control of ThEPC ablation accuracy

(A, B) Lateral views focussed on one ThEPC (green), MIPs of 32 hpf Et(- 1.0otpa:mmGFP)hd1 embryos co-labelled with the thalamic marker lhx2b (red). (C-F) Failed-ablated embryos: Dorsal views, anterior to the left, MIPs of an Et(- 1.0otpa:mmGFP)hd1 embryo at 32 hpf (C) before and (D) after laser treatment (asterisk). Arrowheads highlight the ThEPC cells. (E, F) GFP expression in ThEPC cells recovered 2 hrs after treatment and (E) habenular formation and (F) IPN innervation proceeded normally. (G-I) Dorsal views, anterior to the left, colour coded MIPs of three developmental stages acquired by in vivo 2-PM after cell ablations close to the ThEPC at 32 hpf in an Et(-1.0otpa:mmGFP)hd1 embryo. The LUT shows the Z colour code table according to the depth of each stack. (G, G') White and blue arrowheads show the bilateral location of ThEPC neurons and the second cluster of projecting neurons respectively. The asterisks mark the ablated area next to the ThEPC neurons. (G') The inset shows the position of ThEPC cells before ablation. (H) Yellow dots and arrows highlight the axonal tips of contralateral projecting ThEPC axons and the red dot and arrow marks the tip of a ThEPC axon projecting ipsilaterally. Green arrowheads show the location of the third cluster of projecting neurons. (I) Habenular network architecture at 5 dpf. The IPN is normally innervated by dHb axons. (J, K, M, N) Dorsal views, anterior to the left, MIP of Et(- 1.0otpa:mmGFP)hd1 embryos after complete unilateral dHb cell ablation. (J, M) Asterisks mark the site of ablation, anterior white arrowheads mark non-ablated dHb cells and posterior arrowheads mark ThEPC neurons. (K, N) White arrowheads mark habenula efferent projections entering the IPN on the non-ablated side. Original stacks were cropped and the gamma was adjusted to 0.80 for display purposes. (L, O) 3D-reconstructions of dHb efferent projections innervating the IPN at 4 dpf from dorsal (top) and lateral (bottom). White arrowheads highlight dHb efferent projections entering (L) the ventral IPN after ldHb cell ablation and (O) the dorsal IPN after rdHb cell ablation. Note the characteristic crown shaped structure of dIPN innervating axons in O. d, dorsal; Hb, habenula; IPN, interpeduncular nucleus; l, left; r, right; Tec, optic tectum; ThEPC, thalamic-epithalamic early projecting cluster; v, ventral.