Volume 27, Issue 2, Pages 270-278 (January 2017) Early Commissural Diencephalic Neurons Control Habenular Axon Extension and Targeting  Carlo A. Beretta, Nicolas Dross, Luca Guglielmi, Peter Bankhead, Marina Soulika, Jose A. Gutierrez-Triana, Alessio Paolini, Lucia Poggi, Julien Falk, Soojin Ryu, Marika Kapsimali, Ulrike Engel, Matthias Carl  Current Biology  Volume 27, Issue 2, Pages 270-278 (January 2017) DOI: 10.1016/j.cub.2016.11.038 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Long-Term 2-PM In Vivo Recording Identifies Two Intersected Neural Circuits (A–F) Dorsal view, anterior to the left, color-coded MIP, of six developmental stages acquired by in vivo 2-PM of Et(−1.0otpa:mmGFP)hd1 transgenic embryos. The LUT shows the z color-coded table according to the depth of each slice. The experiment time (hr:min:s) is shown in the left upper corner. Original stacks were cropped and gamma was adjusted to a value of 0.45 for display purposes. The head morphology has been roughly outlined in (A) for orientation. (A and B) White arrowheads mark the ThEPCs at (A) 32 hpf and (B) 44.5 hpf; blue and green arrowheads mark two posterior bilateral clusters of projecting neurons. Red and yellow dots and directional arrows mark the tips of ThEPC axons projecting ipsi- and contralaterally, respectively. Blue and green dots and directional arrows highlight tips of axons from the second and third cluster of contralaterally projecting neurons, respectively. (C) White arrowheads highlight the bilateral expression of GFP in dHb neurons and navigating dHb efferent axons at 49 hpf. (D and E) White arrowheads highlight dHb efferent projections (D) navigating toward the midline before IPN innervation at 53 hpf and (E) thereafter at 65 hpf. (F) Architecture of the habenular neural circuit at 5 dpf. (G) Summary of events during habenular neural circuit development between (top to bottom) 32 and 44.5 hpf, 32 and 53 hpf, and 32 hpf and 5 dpf. ThEPCs, purple; Tec, orange; second cluster of projection neurons, light blue; third cluster of projection neurons, green; ldHb/rdHb and axonal targets, blue/red. Some ThEPC neurons form the vHb [11]. Therefore, the lvHb/rvHb and axonal targets are shown in purple. Note that the schemes do not show the exact number or position of all axons. (H and I) Dorsal views, anterior to the left, focused on early ThEPC neural network formation between (H) 34.5 and 42 hpf and (I) 36 and 42 hpf. The dotted line indicates the embryonic midline. Yellow and red dots highlight the tips of a commissural and an ipsilaterally projecting ThEPC axon, respectively. Yellow and red arrowheads mark the corresponding projection neuron. MIPs were adjusted using the difference of Gaussians to better visualize the structure of interest. d, dorsal; Hb, habenula; Hbl, lateral habenula; Hbm, medial habenula; IPN, interpeduncular nucleus; l, left; LUT, lookup table; MR, median raphe; ob, olfactory bulb; P, pineal; pp, parapineal; r, right; Tec, optic tectum; ThEPC, thalamic-epithalamic early projecting cluster; v, ventral. See also Figures S1 and S2 and Movies S1 and S2. Current Biology 2017 27, 270-278DOI: (10.1016/j.cub.2016.11.038) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 ThEPC Neurons Influence Initiation and Velocity of dHb Axon Elongation (A–B″) Dorsal views, anterior to the left, MIPs of two developmental stages acquired by in vivo 2-PM microscopy in (A and A′) non-ablated and (B and B′) left ThEPC-ablated Et(−1.0otpa:mmGFP)hd1 embryos. An asterisk marks the site of ablation. MIPs were adjusted using the difference of Gaussians to better visualize the structure of interest. The approximate location of the developing IPN is encircled. Yellow arrowheads highlight the position of dHb neurons. The original stacks were cropped and gamma was adjusted to a value of 0.60 for display purposes. Arrows on the right (R) and left (L) side indicate positions chosen for dHb axon growth measures. At positions xR and xL, dHb axons first emerge from the ThEPC in non-ablated embryos and on the non-ablated side in ablated embryos (not shown). aR and aL are the second position, which corresponds to the location of axon tips on the non-ablated side when axons on the ablated side reach xL. bR and bL are the third position, at which dHb axons stall in ablated embryos. VR, VL, VR1, VL1, VRT, and VLT symbolize axon elongation velocities in six (A″) non-ablated and (B″) ablated embryos. VLT and VRT were calculated by dividing the elongation distance of dHb efferent axons and time: (ΔxLbL)/Δt and (ΔxRbR)/Δt with VR1 or VL1 = (ΔxRaR or ΔxLaL)/Δt and VR2 or VL2 = (ΔaRbR or ΔaLbL)/Δt (see Table S1). The error bars represent the SD values of each velocity (V1, V2, VT). Note that measures were carried out in 2D projections and elongation velocity values are arbitrary. (C) Representative example of a 2D dHb axon-tracking plot generated using manual tracking data. Right axons are shown in the upper plot in non-ablated and ablated embryos, whereas the corresponding left axons are shown in the lower plot. Unilateral ablation was performed on the left. Larger and lighter green dots correspond to greater velocity. The right side bar shows the color-coded velocity in μm/hr. dHb, dorsal habenula; l, left; r, right; ThEPC, thalamic-epithalamic early projecting cluster. See also Figures S2–S4, Table S1, and Movie S3. Current Biology 2017 27, 270-278DOI: (10.1016/j.cub.2016.11.038) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Unilateral ThEPC Cell Ablation Causes the Arrest of dHb Efferent Axons on Both Brain Hemispheres (A–F) Dorsal view, anterior to the left, color-coded MIP of six developmental stages acquired by in vivo 2-PM after complete unilateral ThEPC ablation at 32 hpf in an Et(−1.0otpa:mmGFP)hd1 transgenic embryo. Left: the LUT shows the z color-coded table according to the depth of each slice. Original stacks were cropped and gamma was adjusted to a value of 0.60 for display purposes. The head morphology has been roughly outlined in (A) for orientation. (A) The yellow dot and arrow mark the tip of a ThEPC axon. The blue arrowheads highlight the position of the second cluster of commissural neurons. (B) The yellow dots and arrows mark the tips of axons starting to migrate toward the midline, but which turn subsequently. The red dot and arrowhead label the tip of an ipsilateral ThEPC axon. The green arrowheads highlight the position of the third cluster of commissural neurons. (C) White arrowheads show the bilateral expression of GFP in the dHb. A white square highlights the area in which dHb axon elongation stalls. Note the delay of dHb axons on the left side. (D and E) White arrowheads highlight the position of extending dHb efferent axons at (D) 68 hpf and (E) 74 hpf. (F) Architecture of the habenular neural circuit after left-sided ThEPC cell ablation at 5 dpf. White arrowheads mark the ends of the stalled dHb efferent axon bundles. (G) Summary of events during habenular neural circuit development after complete unilateral ThEPC ablation between (top to bottom) 42 and 52 hpf, 42 and 68 hpf, and 42 hpf and 5 dpf. ThEPCs and rvHb, purple; Tec, orange; second cluster of projection neurons, light blue; third cluster of projection neurons, green; ldHb/rdHb and axonal projections, blue/red. (H) Dorsal view, anterior to the left, MIPs of eight developmental stages focused on a turning commissural ThEPC axon on the non-ablated site. The axon’s tip is marked by a yellow dot and its neuron with a yellow arrowhead. Segmented images show events between 34 and 43 hpf. MIPs were adjusted using the difference of Gaussians to better visualize the structure of interest. Asterisks mark the site of ThEPC ablation between 32 hpf and 5 dpf. d, dorsal; Hbl, lateral habenula; Hbm, medial habenula; IPN, interpeduncular nucleus; l, left; MR, median raphe; ob, olfactory bulb; r, right; Tec, optic tectum; ThEPC, thalamic-epithalamic early projecting cluster; v, ventral. See also Figures S3 and S4 and Movie S3. Current Biology 2017 27, 270-278DOI: (10.1016/j.cub.2016.11.038) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Complete and Incomplete Unilateral ThEPC Cell Ablations Cause Differently Severe Bilateral IPN Innervation Defects (A–D) Dorsal view, anterior to the left, color-coded MIP of four developmental stages acquired by in vivo 2-PM after sequential unilateral ThEPC cell ablation at 32 hpf in an Et(−1.0otpa:mmGFP)hd1 embryo. The LUT shows the z color-coded table according to the depth of each stack. Original stacks were cropped and gamma was adjusted to a value of 0.60 for display purposes. (A) For orientation, white arrowheads mark the position of ThEPC neurons; blue arrowheads mark the second posterior bilateral cluster of projecting neurons. An asterisk marks the ablated side. (B) White arrowheads mark the left and right dHb. The blue dot and arrow mark the tip of a commissural tectal axon; red and yellow dots and arrows mark ipsilateral and commissural ThEPC axons, respectively. (C and D) White arrowheads and box indicate the area in which dHb axon elongation stalls upon complete ThEPC cell ablation at (C) 66 hpf and (D) 119 hpf. (E and F) Statistics of IPN innervation phenotypes observed after (E) 74 independent unilateral complete and (F) 28 sequential ThEPC cell ablations. Numbers above the columns correspond to the number of embryos showing the respective phenotype. (G–I) Dorsal views with anterior to the left showing normal IPN innervation (G) and examples of the indicated IPN innervation phenotypes after sequential unilateral ThEPC neuron ablations (H and I). Asterisks in (H) and (I) mark the site of ablation. d, dorsal; Hb, habenula; IPN, interpeduncular nucleus; l, left; r, right; Tec, optic tectum; ThEPC, thalamic-epithalamic early projecting cluster. See also Figures S3 and S4 and Movie S3. Current Biology 2017 27, 270-278DOI: (10.1016/j.cub.2016.11.038) Copyright © 2017 Elsevier Ltd Terms and Conditions