Fig. 6

The telencephalic vENT is a major source of habenula afferents, is diencephalic in origin and originates from the prethalamic eminence. The vENT projects to the habenula. (A) Schematic summarizing experimental setup. Habenula neuropil of 72 hpf Tg(lhx5:Kaede)^b1204 was photoconverted with a cone of UV light. Photoconversion was made with a lateral approach in order to avoid conversion of areas ventral to the habenula. (B,B′) Lateral views of single Z-slices of Tg (lhx5:Kaede)^b1204 larvae (B′ is closer to the midline) imaged at 4dpf after photoconversion of the habenula neuropil at 72 hpf. Dotted line marks the anterior intraencephalic sulcus. Some red Kaede cells (magenta in the figure) are observed in the olfactory bulb (OB) and preoptic region (PO), and a few fibers in Dp. A number of red Kaede cells were observed in the domain identified as presumptive larval vENT (arrow in B′). Note red Kaede fibers at the photoconversion area in the habenula (arrowhead in B). The vENT expresses tbr1 and straddles the telencephalic/diencephalic boundary at 4dpf. (C-C′′) Lateral views of Tg(lhx5:GFP)^b1205 inmunostaining against GFP (green) and FISH against tbr1 (grayscale). In C, the vENT has been false colored in pink. Dotted lines on lateral views mark the anterior intraencephalic sulcus, which runs from the optic recess at the base of the optic stalks ventrally to the pallial/diencephalic boundary dorsally. (C) 3D projection of a confocal stack showing tbr1 and GFP co-expression in the putative vENT (false colored in pink to differentiate from pallial and subpallial tbr1a expression). (C′,C′′) Single confocal z-slices at lateral (C′) and medial levels (C′′) showing GFP and tbr1 coexpression in vENT neurons (arrow). Note that the vENT extends from the diencephalon into the telencephalic lobes (dotted line marks the anterior intraencephalic sulcus). At medial levels, the larval vENT surrounds the lateral forebrain bundle (lfb, star in C′′), as has been observed in the adult (see Figure 1F). The vENT is diencephalic in origin and originates from the prethalamic eminence. Lateral (D,E,G,H) and transverse views (F,I) of Tg(lhx5:GFP)^b1205 fish labeled with anti-GFP (green) and FISH for tbr1 (pink) (D-D′,G,G′) or dlx1a (blue) (E,F,H,I) at 48 hpf (D-F) and 72 hpf (G-I). All images are single confocal z-slices taken from volumes, anterior to the left. (D-F) Lateral (D,E) and transverse view after 3D rendering (F) of Tg(lhx5:GFP)^b1205 48 hpf larva labeled with anti-GFP (green) and FISH for tbr1+ (pink in D) and dlx1a+ (blue in E,F). Dotted yellow line in (D,E) marks the dorsal limit of the tbr1+ domain. (D) A group of GFP and tbr1+ cells (arrow) are situated in the diencephalon, just caudal to the telencephalic/diencephalic boundary. These cells were identified as prethalamic eminence/prospective vENT. (D′) Same image as (D) but only showing GFP expression (green channel). Arrow points to prethalamic eminence/prospective vENT. (E) GFP and dlx1a+ cells in the prethalamus, just dorsal to the prethalamic eminence/prospective vENT (arrow). (F) Transverse section shows the relative mediolateral position of the prethalamus, vENT and preoptic areas. (G-I) Lateral (G,H) and transverse section (I) of Tg(lhx5:GFP)^b1205 72 hpf larva labeled with anti-GFP (green) and FISH for tbr1+ (pink in G) and dlx1a+ (blue in H and I). Dotted yellow line in (D,E) marks the dorsal limit of the tbr1+ domain. (G) The GFP+ vENT expresses tbr1 extends from the ventrolateral telencephalon to the rostral diencephalon (arrow points to the rostral portion of the vENT, dotted yellow line marks the dorsal limit of the tbr1+ domain). (G′) Image shown in (G) showing just GFP expression (green channel). (H) dlx1a+ domain in the prethalamus (PTh), just dorsal to the diencephalic portion of the vENT (arrow points to rostral portion of the vENT, dotted yellow line marks the dorsal limit of the tbr1+ domain in the diencephalon). (I) Transverse section shows the GFP+ vENT (green) laterally located and dlx1a+ PO (blue) areas more medially. (J) Schematic representations of the brain of 48 and 72 hpf larvae showing expression of lhx5:GFP, dlx1a and tbr1 in the prethalamus (PTh), prethalamic eminence (arrow) and preoptic region (PO). Time-series showing the development of the prethalamic eminence that gives rise to the vENT. (K) Time series showing tbr1 expression from 24 to 72 hpf in Tg(lhx5:GFP)^b1205. (i) At 24 hpf, there is no tbr1 expression in rostral diencephalon; (ii) At 36 hpf brain, tbr1 expression is observed in a subset of the GFP+ cells in the diencephalon (arrow) close to the the telencephalic/diencephalic boundary (marked by dotted line). These cells are likely to be within the prethalamic eminence/prospective vENT; (iii-v) From 48-72 hpf, vENT cells coexpressing tbr1 and GFP extend into the telencephalic lobes (arrow points to rostral portion of the vENT). Scale bars: (B,C,D′,G′) 50 µm; (K) 100 µm.