Figure 1.

Identification of DDNs in the Diencephalon of ETvmat2:GFP Zebrafish Larvae at 4 dpf

(A) Ventral (left) and lateral (right) schematic overview of DC2 (dark green) and DC4/5 (light green) neurons in the diencephalon (dashed line represents the anterior diencephalic border) of 4 dpf larvae.

(B) Ventral view of GFP-labeled cells in the diencephalon. Prospective DDNs in DC2 comprise a small cluster of strongly fluorescent, large-diameter cells (dashed boxes) near the anterior aspect of the diencephalon.

(C) Lateral images of the posterior tuberculum labeled with GFP (green, top) and anti-tyrosine hydroxylase antibodies (Anti-TH, yellow, middle). Merged image (bottom) reveals that all large, GFP-positive neurons in the anterior aspect of posterior tuberculum are immunoreactive for TH.

(D) Lateral view of a GFP-expressing neuron in the anterior posterior tuberculum (green) labeled with neurobiotin (NB, red) and co-stained with anti-tyrosine hydroxylase antibodies (Anti-TH, yellow). Merged image (bottom right) reveals NB-labeled cells are GFP and TH positive.

(E and F) Lower-magnification contrast inverted composite images of the same NB-labeled neuron in (D) depicting the soma and primary axon in the diencephalon (E; arrows) and the primary axon (F; arrows) extending from the hindbrain (HB) to the spinal cord (SC). Extensive axonal arborization is observed near the soma (arrow heads in E).

(G and H) At the level of the hindbrain, the primary axon branches into the periphery, innervating the otic capsule (OC, top in G), cranial neuromasts (CNM, bottom in G), lateral line (top in H), and trunk neuromasts (TNM, bottom in H). White dashed lines in (G) and (H) mark the OC (top) and CNM and TNM (bottom). Bottom panels in (G) and (H) depict contrast-inverted image of NB labeling (left), bright field (middle), and merged NB-bright field images (right).

(I and J) Ventral (I) and lateral (J) schematic overviews of DDNs and their arborization patterns. Axons project caudally into the spinal cord (solid green lines) and into the periphery (dashed green lines).

In (B)–(H), anterior is left and posterior is right. In (C)–(H), dorsal is up and ventral is down. Scale bars in (B)–(H) represent 10 μm. (A), (I), and (J) are not to scale.