PMC

Timeline showing the ontogeny of Cx35 immunoreactivity in the CNS of zebrafish from 1 dpf through 15 dpf. Abbreviations are as given in the text.

Cx35 immunoreactivity in Mauthner neurons of 6 dpf larvae. The left panels show retrograde labeling of a Mauthner neuron with tetra-methyl rhodamine dextran (TMR dextran, red). The middle panels show Cx35 immunoreactivity in green and the right panels show merge of the two channels. All images are single optical sections taken at a z-spacing of 0.44 μm. (A) Cx35-immunoreactive puncta line the Mauthner axon and form a dense cloud in the axon-cap region (arrowhead). (B) Club endings on the lateral dendrite are large bouton-like structures (arrowhead) that are intensely Cx35 immunoreactive. (C) Cx35 puncta are also seen to line the Mauthner soma. Scale bar for all panels: 20 μm.

Dye-coupled cells in the hindbrain of 4 dpf larvae. Larvae are dorso-ventrally placed with anterior on top in all panels. All panels show single optical sections taken at a z-resolution of 0.44 μm. (A–C) Images at depths of 20, 30, and 43 μm from the ventral surface showing reticulospinal neurons retrogradely labeled with neurobiotin (green) and tetramethyl rhodamine dextran (red). Cells electrically coupled to the labeled neurons but not projecting into the spinal cord are green in color, while the neurons that project into the spinal cord are yellow because of the presence of both neurobiotin and tetra methyl rhodamine dextran. Scale bar for all panels: 20 μm.

Cx35 staining pattern in 1 dpf larva. (A) Single optical section of the dorsal pallium (Pd). (B) Optic tectum (OT). (C) Hindbrain (Hb). The cerebellum (CCe), and the rhombencephalic ventricle (RV) can be also be seen. Anterior is at the top and posterior is at the bottom. Larva is in dorsoventral position. (D–F) Single optical sections from control larvae in which the primary antibody was not included in the incubation mixture. (D) Dorsal telencephalon. (E) Optic tectum. (F) Hindbrain. (G–I) Transmitted light images of the regions imaged in (D–F). Scale bar for all panels: 20 μm.

Cx35 staining pattern in the CNS of 15 dpf zebrafish. Larvae are dorso-ventrally placed with anterior on top in all panels. All panels show single optical sections taken at a z-resolution of 0.44 μm. (A) Ventral telencephalon showing punctate Cx35 immunoreactivity in the anterior commissure (AC) and the olfactory bulb (OB). (B) Telencephalon 50 μm from the ventral surface showing staining in the anterior commissure (AC) and in the olfactory bulb (OB). The telencephalic ventricle (TelV) can be seen. (C) Mesencephalon, dorsal view showing staining in the optic tectal (OT) neuropil and in intertectal commissures (Ctec, arrowhead). (D) Habenulae (Hab) and the habenular commissure (Chab; arrowhead). Polygonal cell bodies can be seen in the habenulae (E) Cerebellum showing intense Cx35 immunoreactivity in the corpus cerebelli (CCe) and the molecular layer (ML) (F) Ventral Hindbrain (Hb) showing a ladder-like pattern that includes Cx35 staining in the lateral longitudinal fasciculus (LLF) and hindbrain commissures. The rhombencephalic ventricle (RV) can be seen. (G) Intense punctate Cx35 immunoreactivity in the Mauthner neurons (M; arrowheads). The approximate boundaries of the Mauthner neurons is drawn in yellow. Scale bar for all panels: 20 μm.

Cx35 distribution in the CNS of 2 dpf larvae. Larvae are dorso-ventrally placed with anterior on top in all panels. All panels except (F) show single optical sections taken at a z-resolution of 0.44 μm. (A) Ventral telencephalon showing the anterior commissure (AC), supra-optic tract (SOT), and the post-optic commissure (POC). (B) Dorsal view of forebrain showing Cx35 positive cells in the dorsal pallium (Pd). The posterior commissure can be seen (arrowhead) while the habenulae (Hab) and the pineal body (P) lack Cx35 immunoreactivity. (C) Dorsal view of the optic tectum (OT). A single optical section showing Cx35 immunoreactivity in the developing neuropil region (arrowheads). (D) Cell bodies (arrowheads) in hindbrain lining the rhombencephalic ventricle. (E) Ventral hindbrain (Hb) showing intense Cx35 immunoreactivity in fibers coursing through it. (F) Zoomed image of a Mauthner neuron (M, yellow outline) showing punctate Cx35 immunoreactivity. The image is a maximum intensity projection of optical slices collected from a 16 μm thick tissue slice encompassing the Mauthner neuron. Scale bar for all panels: 20 μm.

Distribution of Cx35 immunoreactivity in 3 dpf larvae. All panels except (C) show dorso-ventrally placed larvae, with the anterior end of the animal at the top of the panel. All panels are single optical sections taken at a z-resolution of 0.44 μm. (A) Ventral forebrain showing immunoreactivity in the anterior commissure (AC) and in the tract of the post-optic commissure (TPOC). (B) Dorsal telencephalon and rostral mesencephalon. Staining in cells of the dorsal pallium (Pd) and in the posterior commissure (PC) can be seen. (C) Lateral view of forebrain. Dorsal is on top and anterior to the left. Cx35 immunoreactivity is seen in the anterior commissure, in the supra-optic tract and in thick fiber tracts in ventral diencephalon. Numerous cell bodies can also be seen in the vicinity of the anterior commissure. (D) Intense Cx35 immunoreactivity in the optic tectal (OT) neuropil. (E) Ventral hindbrain (Hb) showing fiber tracts criss-crossing the hindbrain. (F) Zoomed image of a single Mauthner neuron (M) showing intense Cx35 immunoreactive puncta. The approximate boundary of the Mauthner neuron as seen in DIC is shown in yellow. Scale bar for all panels: 20 μm.

Cx35 immunostaining in 4 dpf larvae. Larvae are dorso-ventrally placed with anterior on top in all panels. All panels show single optical sections taken at a z-resolution of 0.44 μm. (A) Dorsal forebrain showing Cx35 staining in the paired habenulae (Hab) and in the habenular commissure (Chab). Cell bodies in dorsal pallium (Pd) continue to stain positive. No staining is seen in the pineal body (P). (B) Ventral telencephalon showing Cx35 immunoreactivity in the olfactory bulb (OB), anterior commissure (AC), supra-optic tract (SOT), post-optic commissure (POC), and the tract of the post-optic commissure (TPOC). (C) Cx35 immunoreactivity in fiber tracts in the ventral diencephalon (Di). (D) Intense Cx35 immunoreactivity in the optic tectal neuropil (OT) and in the intertectal commissures (Ctec; arrowhead) in the mesencephalon. Tectal cell bodies are also weakly labeled. (E) Weak staining is seen in the cerebellum within the molecular layer (ML) and in the corpus cerebelli (CCe). (F) Zoomed image of a single Mauthner neuron (M) showing intense Cx35 immunoreactive puncta. The approximate boundary of the Mauthner neuron as seen in DIC is shown in yellow. Scale bar for all panels: 20 μm.

Distribution of Cx35 immunoreactivity in the CNS of 6 dpf larvae. Larvae are dorso-ventrally placed with anterior on top in all panels. All panels show single optical sections taken at a z-resolution of 0.44 μm. (A) Ventral telencephalon showing staining in anterior commissure (AC) and the olfactory bulb (OB). A few faintly stained cell bodies can be seen in the vicinity of the anterior commissure. (B) Dorsal forebrain showing Cx35 staining in the habenulae (Hab), dorsal pallium (Pd), and the posterior commissure (PC; arrowhead). (C) Magnified dorsal view of the optic tectum (OT) showing intense staining in the neuropil area. Faint labeling in cell bodies is also seen (arrowhead). (D) Cx35 staining in the mesencephalon showing intense staining in the optic tectal lobes in the neuropil area as well as in intertectal commissures (Ctec; arrowhead). The corpus cerebelli (CCe) can also be seen. (E) Cerebellar staining showing punctate pattern within the corpus cerebelli (CCe) and the molecular layer (ML). (F) Strong staining present in radial glia (RG) lining the rhombencephalic ventricle. Arrowhead marks the process from one glial cell coursing laterally. (G) Ladder-like staining pattern in ventral hindbrain (Hb). Scale bar for all panels: 20 μm.