dcc loss-of-function generates an ectopic axon bundle in the diencephalon. All images are confocal projections of zebrafish whole-mounted brains at 30 hpf immunolabelled for expression of acetylated α-tubulin. Anterior is left in all images. (A) The axon tracts are displayed according to Fig. 1. Note the absence of axons in the diencephalic territory between the DVDT and DRC–SOT except for a few pioneers of the future THC (large arrow). (B) Note the absence of any axon tract in the diencephalic space separating the DRC from the EC. The dotted line represents the midline. (C) Axons emerging from the posterior edge of the DRC and extending into the diencephalon are indicated by the thin arrows. These axons typically follow a curved route through the diencephalon down to the TPOC, meeting with the pioneers of the THC extending dorsally or the DVDT (large arrow). (D) The ectopic axons clearly extend from the caudal margins of the DRC into the dorsal diencephalon. Most axons are tightly bundled at the exit of the telencephalic cluster. (E) Co-knockdown of shh and dcc did not rescue the phenotype of these ectopic diencephalic axons nor did it alter their route ventrally (arrow) since they still connect with the TPOC (large arrow). (F) While the left side shows a typical tightly-fasciculated bundle of ectopic axons (arrow), the right side of this embryo exhibits axons wandering individually in the dorsal diencephalon (arrowheads). These individual axons appear to be still following a stereotypical route as in the contralateral side of the brain. Scale bar=35 μm (A, C, E), 20 μm (B, D, F).

Knocking down ntn1a, but not ntn1b, reproduces the dcc loss of function phenotype. All images are confocal projections of zebrafish whole-mounted brains at 30 hpf immunolabelled for expression of acetylated α-tubulin. (A) Ectopic axons emerge from the caudal edge of the DRC (thin arrow). The large arrow indicates THC pioneer axons that extend from the ventral diencephalon. (B) Ectopic axons emerge caudally from the DRC. In this example, the tract emerging from the right side of the telencephalon crosses the midline (arrowhead) and invades the contralateral diencephalon before joining the contralateral ectopic axons and continuing the stereotypical curved route to the ventral part of the diencephalon (thin arrow). (C) No ectopic axons are visible in the diencephalic territory caudal to the DRC except for the ventrally located pioneers of the THC (large arrow). Note the axons ventral to the AC that seem to detach themselves from the bulk of the commissure (thin arrows). (D) No ectopic axons are observed in the dorsal diencephalon. (E) The ectopic axon tract follows a stereotypical course through the diencephalon (thin arrow). (F) Ectopic axons (arrows) do not always cross the midline. Scale bars=40 μm (A, C, E), 20 μm (B, D, F).

EDAB is distinct from the stria medullaris. All images are confocal projections of zebrafish whole-mounted brains immunolabelled for expression of acetylated α-tubulin. (A), (B), (D) Lateral view (anterior is left, dorsal is up). (C) Dorsal view (anterior is left). (A) At 36 hpf, two new axon tracts extend into the diencephalon: the tract of the habenular commissure (large arrow) and the pioneer axons of the stria medullaris (chevron arrow). (B) At 48 hpf, the stria medullaris (chevron arrow) and the tract of the habenular commissure (large arrow) have both enlarged considerably and have fused together to project to the habenula in the dorsal diencephalon. (C) Dorsal view of a 48 hpf dcc+ntn1a MO injected brain exhibiting the typical ectopic diencephalic axon tract in the dorsal diencephalon (thin arrow). Note that the left-side ectopic tract crosses the midline (arrowhead) before merging with its contralateral counterpart. The ectopic tract that remained ipsilateral merged with the THC (large arrow). Chevron arrows indicate the stria medullaris on both sides. (D) Lateral view of a 48 hpf dcc+ntn1a MO injected brain. Note the dorsal ectopic tract (thin arrow) that is distinct from the stria medullaris (chevron arrow) and fusing into the THC (large arrow). Scale bars=40 μm (A, B, D), 30 μm (C).

EDAB originates from a subset of neurons in the caudal portion of the DRC that normally project into the anterior commissure. Axon tracing using kaede photoconversion (A–D), chimeras (E–F) and DiI (G–L′). All images are confocal image stacks processed with Imaris software. The resulting representation are either a simple maximum intensity projection under orthogonal perspective along the Z axis (E–K), perspective views of shadow projection rendering of the stacks (A–D, K′, L′) or a mix of shadow projection rendering and volume rendering (F). (A) Dorsal view of the DRC in a dcc knock down embryo at 30 hpf. When the anterior portion of the DRC was photoconverted (red), the EDAB remained green (arrowheads) while red photoconverted axons exited via the SOT (large arrow). (B) Dorsal view of the DRC in a dcc knock down embryo at 30 hpf. When the posterior portion of the DRC was photoconverted (red) the EDAB was red (arrowheads). Note also the large number of photoconverted axons extending towards the anterior commissure (chevron). (C) Lateral view of a control brain at 30 hpf. The dotted line indicates the path of the photoconverting laser through the DRC and the underlying VRC. When the posterior portion of the DRC is photoconverted (red) in a control brain, both the anterior commissure (chevron arrow) and SOT (large arrow) are labelled in red. (D) Lateral view of a dcc knock down embryo at 30 hpf. The dotted line represents the path of the photoconverting laser through the DRC and the caudal VRC and TPOC. The EDAB (arrowhead), anterior commissure axons (chevron arrow) and SOT axons (large arrow) are all labelled in red. (E) Lateral view of the DRC in a wild-type host chimeric animal immunolabelled for expression of acetylated α-tubulin (red) showing the axon trajectory of donor neurons expressing kaede from the caudal edge of the DRC (large arrows) into the anterior commissure (arrowheads). (F) Lateral view of the forebrain of a wild-type host chimeric animal immunolabelled for expression of actetylated α-tubulin (red) showing growth cones of kaede-expressing donor cells (large arrow) in the anterior commissure tract (arrowhead) and the SOT (thin arrow), representing the two routes used by axons originating from the caudal half of the DRC. (G) Lateral view of the forebrain of a dcc knock down embryo showing the site of microiontophoretic deposit of DiI in the route of the EDAB (large arrow). The EDAB axon is anterogradely labelled (thin arrow). Retrograde labelling reveals the cell body of the neuron located at the dorsocaudal end of the DRC (arrowhead). (H)–(J) Lateral views showing the localisation of the cell bodies giving rise to the EDAB in the caudal half of the DRC (arrowheads) after retrograde DiI labelling of the EDAB. (K)–(K)′ Lateral view of the DRC in a control embryo after injection of DiI in the anterior commissure (large arrow). Axons are retrogradely labelling the cell bodies located at the dorsocaudal end of the DRC (arrowhead). (L)–(L)′ Lateral view of the DRC in a control embryo after injection of the DiI at the dorsocaudal end of the DRC (large arrow) labelling axons anterogradely in the tract of the anterior commissure. These axons enter the anterior commissure after passing the point where the SOT bifurcates from the tract. Scale bars=15 μm (A–D), 5 μm (E), 10 μm (F), 20 μm (G), 25 μm (H–J), 10 μm (K–L′).

Tracing the EDAB to the ventrorostral diencephalic cluster. (A) HuC:kaede brain. (B) HuC:kaede/Foxd3:GFP brain. DiI labelling is represented in red and the kaede/GFP fluorescence in green. The star indicates the site of DiI injection, the arrowhead indicates the anterior commissure, the large arrow is the supraoptic tract and the thin arrow points at the identifiable end of the EDAB. In (B) the DVDT trajectory is clearly depicted by GFP fluorescence in green. Scale bar=50 μm.

Acknowledgments
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Reprinted from Developmental Biology, 367(2), Gaudin, A., Hofmeister, W., and Key, B., Chemoattractant axon guidance cues regulate de novo axon trajectories in the embryonic forebrain of zebrafish, 126-139, Copyright (2012) with permission from Elsevier. Full text @ Dev. Biol.