Jip3, an actively transported protein, was necessary for axon extension and the prevention of axon terminal swellings.
(A) Schematic of a larval zebrafish illustrating the basic anatomy of the primary posterior lateral line (pLL) system. Neuromasts (NMs; terminal NM cluster-ter) are innervated by the pLL nerve (green), which emanates from the pLL ganglion (pLLg). (B) Wildtype neurod:EGFP transgenic at 5 dpf with the pLLg and pLL nerve (pLLn) indicated. (B′,B′′) Panels illustrate pLL axon terminals that innervate NM3 (B′) and the distal end of the pLL nerve including the axon terminals at the terminal NM cluster (ter; B′′; red arrowheads point to axon terminals). (C) jip3nl7 mutants displayed truncated pLL nerves and distal pLL nerve thinning (C′′) as well as swollen axon terminals in all NMs (NM3 shown in C2). Scale bars B and C = 100 μm. Scale bars in B′, B′′, C′ and C′′ = 10 μm. (D, E) Long central nervous system axons of the reticulospinal tract (arrowhead) and pLL efferent axons (arrow), visualized by the phox2b:EGFP transgenic reporter, were also truncated in jip3nl7 mutants. End of trunk indicated by the asterisk. (F) Schematic of the zebrafish Jip3 protein showing conserved structural and binding domains. The red arrowhead indicates the location of the jip3nl7 mutation, which generates a premature stop codon at amino acid 184. (G,H) In situ hybridization analysis revealed that jip3 was expressed in the central and peripheral nervous systems at 2 dpf in wildtype but was lost in jip3nl7. (I) Schematic of the paradigm designed to image axon transport in the pLL nerve. (J) Transient expression of Jip3-mCherry in 1 neuron of the pLL ganglion at 30 hpf. (K) Jip3-mCherry was localized to a growth cone of an extending axon at 30 hpf. The pLL ganglion and nerve were visualized by expression of the neurod:EGFP transgene. (L) Jip3-mCherry is actively transported in pLL axons (Video S1). Arrowhead (pink) and arrow (yellow) indicate anterograde and retrograde particle movement respectively. (M) Kymograph of time-lapse imaging in J. Scale bars in J–L = 10 μm.