JNK-Interacting Protein 3 Mediates the Retrograde Transport of Activated c-Jun N-Terminal Kinase and Lysosomes

<div><p>Retrograde axonal transport requires an intricate interaction between the dynein motor and its cargo. What mediates this interaction is largely unknown. Using forward genetics and a novel <i>in vivo</i> imaging approach, we identified JNK-interacting protein 3 (Jip3) as a direct mediator of dynein-based retrograde transport of activated (phosphorylated) c-Jun N-terminal Kinase (JNK) and lysosomes. Zebrafish <i>jip3</i> mutants (<i>jip3^nl7</i>) displayed large axon terminal swellings that contained high levels of activated JNK and lysosomes, but not other retrograde cargos such as late endosomes and autophagosomes. Using <i>in vivo</i> analysis of axonal transport, we demonstrated that the terminal accumulations of activated JNK and lysosomes were due to a decreased frequency of retrograde movement of these cargos in <i>jip3^nl7</i>, whereas anterograde transport was largely unaffected. Through rescue experiments with Jip3 engineered to lack the JNK binding domain and exogenous expression of constitutively active JNK, we further showed that loss of Jip3–JNK interaction underlies deficits in pJNK retrograde transport, which subsequently caused axon terminal swellings but not lysosome accumulation. Lysosome accumulation, rather, resulted from loss of lysosome association with dynein light intermediate chain (dynein accessory protein) in <i>jip3^nl7</i>, as demonstrated by our co-transport analyses. Thus, our results demonstrate that Jip3 is necessary for the retrograde transport of two distinct cargos, active JNK and lysosomes. Furthermore, our data provide strong evidence that Jip3 in fact serves as an adapter protein linking these cargos to dynein.</p> </div

Fgf3 and Fgf10a Work in Concert to Promote Maturation of the Epibranchial Placodes in Zebrafish

<div><p>Essential cellular components of the paired sensory organs of the vertebrate head are derived from transient thickenings of embryonic ectoderm known as cranial placodes. The epibranchial (EB) placodes give rise to sensory neurons of the EB ganglia that are responsible for relaying visceral sensations form the periphery to the central nervous system. Development of EB placodes and subsequent formation of EB ganglia is a multistep process regulated by various extrinsic factors, including fibroblast growth factors (Fgfs). We discovered that two Fgf ligands, Fgf3 and Fgf10a, cooperate to promote EB placode development. Whereas EB placodes are induced in the absence of Fgf3 and Fgf10a, they fail to express placode specific markers Pax2a and Sox3. Expression analysis and mosaic rescue experiments demonstrate that Fgf3 signal is derived from the endoderm, whereas Fgf10a is emitted from the lateral line system and the otic placode. Further analyses revealed that Fgf3 and Fgf10a activities are not required for cell proliferation or survival, but are required for placodal cells to undergo neurogenesis. Based on these data, we conclude that a combined loss of these Fgf factors results in a failure of the EB placode precursors to initiate a transcriptional program needed for maturation and subsequent neurogenesis. These findings highlight the importance and complexity of reiterated Fgf signaling during cranial placode formation and subsequent sensory organ development.</p> </div

Increased levels of pJNK did not cause lysosome accumulation in <i>jip3^nl7</i>.

<p>(A) Induction of caJNK3-EGFP at 4 dpf increased the level of pJNK immunofluorescence (middle) in a subset of axon terminals but did not lead to lysosome accumulation as compared to control (B). Scale bars = 10 µm. (C, D) This result was confirmed by Lysotracker red labeling. Surrounding, non-caJNK3-EGFP positive axons show similar numbers, size and density of lysosomes both 4 hours and 13 hours after induction of caJNK3. The pLL nerve was visualized by phase contrast optics and is outlined. Arrowhead indicates axonal swellings caused by high levels of activated JNK. HC denotes neuromast hair cells that strongly label with Lysotracker red. (E) Whole embryo expression of Jip3 and Jip3ΔJNK by mRNA injection partially suppressed the accumulation of lysosomes in <i>jip3^nl7</i> mutant axon terminals at 3 dpf as assayed by expression of Lamp1-mTangerine in pLL neurons. Wildtype – Lamp1-mTangerine positive small puncta only; Mild – small puncta and aggregates visible; Severe - few to no small puncta apparent and large aggregations of Lamp1-mTangerine. (F–I) Injection of 10 pg of a DNA construct encoding Jip3ΔJNK-mCherry rescued lysosome accumulation in <i>jip3^nl7</i> axon terminals. Larvae that expressed Jip3ΔJNK-mCherry (red) in pLL axons and carried the <i>neurod:EGFP</i> transgene were first imaged live (F,H) to identify expressing axon terminals. They were then individually fixed, stained for pJNK (pseudo-colored magenta) and Lamp1 (white), and subsequently the same axon terminals were reimaged (G,I). Arrowheads point to axon terminals in wildtype (F,G; NM1) and <i>jip3^nl7</i> (H,I; NM5) that express Jip3ΔJNK-mCherry (red) at 5 dpf. Arrows point to axon terminals in the same NMs that did not express this construct. Note that expression of Jip3ΔJNK-mCherry in <i>jip3^nl7</i> completely rescued lysosome accumulation (yellow arrowheads in I″) but failed to rescue high levels of pJNK (yellow arrowheads in I′).</p

Fgf3 and Fgf10a are expressed during epibranchial placode formation.

<p>(<b>A</b>-<b>C</b>) In situ hybridization reveals presence of fgf3 transcript in the mesoderm at 14 hpf (A), and then in the endoderm at 18 (B) and 22 hpf (C). (<b>D</b>-<b>F</b>) In situ hybridization reveals presence of <i>fgf10a</i> transcript in the anterior and posterior lateral line (arrows) and the anterior portion of the otic vesicle at 14 (D),18 (E) and 22 hpf (F). Otic vesicle is outlined by a dotted line in (A-F). Abbreviations: ov, otic vesicle; me, mesoderm; en, endoderm; All, anterior lateral line; Pll, posterior lateral line. Scale bar: 50 µm. </p

pJNK failed to accumulate distal to injury.

<p>(A) Schematic and time-line of the injury model experiment and fluorescent intensity quantification. pLL nerve (identified using the <i>neurod:EGFP</i> transgene) was severed using finely pulled glass capillaries. DIC image of a representative injury illustrates peripheral tissue remained mostly intact. Three hours post-injury, larvae were fixed and stained for GFP (to identify the nerve) and either pJNK or tJNK. Thirty µm areas immediately proximal or distal to the injury were imaged and the mean fluorescent intensity of pJNK or tJNK was determined in summed projected stacks through the nerve only in areas that overlapped with GFP expression (outlined by dotted lines in B–I). Background mean fluorescent intensity was determined in adjacent tissue. (B–I) Proximal and distal nerve (dotted outline) adjacent to site of injury (dashed line) in wildtype and <i>jip3^nl7</i> larvae immunolabled for pJNK (B–E) and tJNK (F–I). (J) Levels of pJNK were decreased distal to nerve injury in <i>jip3^nl7</i> but proximal levels were comparable to wildtype (ANOVA, post-hoc contrasts; *-<i>p</i><0.05). (K) tJNK levels trended towards a decrease proximal to the site of injury in <i>jip3^nl7</i> (ANOVA, post-hoc contrasts; <i>p</i><0.1790) but were not different in the retrograde pool, distal to axonal severing.</p

The anterior lateral line is the tissue source of Fgf10a responsible for facial placode development.

<p>(<b>A</b>-<b>D</b>) confocal projections of Tg(pax2a:EGFP) zebrafish embryos (green) analyzed for Pax2a expression (magenta) at 14 (A), 18 (B), 21 (C), and 24 hpf (D). The presumptive facial placode is outlined in yellow as it condenses between 18 and 24 hpf. Insets show co-expression of Pax2a and Tg(pax2a:EGFP) at 14 hpf (A); by 18 hpf, however, Pax2a expression is absent in anterior lateral line precursors, while Tg(pax2a:EGFP) maintains expression in these cells (B). (<b>E-E</b>’’) Live confocal projection of a 12 hpf Tg(pax2a:Kaede) zebrafish embryo (E) with the anterior portion of the <i>pax2a:Kaede</i>+ domain photoconverted from green to red emission (E’) overlay (E’’). (<b>F-F</b>’’) Composite image of the same photoconverted embryo from (E) at 24 hpf analyzed for Pax2a expression (F) and cyan in (F’’) and photoconverted Kaede (F’) and red in (F’’). Note absence of Kaede positive cells in the facial placode. (<b>G</b>, <b>H</b>) In situ hybridization of <i>eya1</i> in 52 hpf zebrafish embryos reveals proper neuromast deposition in control (G) and a failure of deposition and elongation of the anterior lateral line in fgf3-/-;fgf10-MO embryo (H; arrowheads). (<b>I</b>, <b>J</b>) Lateral views of the 24 hpf fgf3<i>+10</i> morphant embryo showing the side that received wild-type donor cells (green) as well as the contralateral control side that did not receive donor cells. Pax2a expression is visualized by immunolabeling (magenta). Note partial rescue of the facial placode when wild-type donor cells were present in the presumptive anterior lateral line (J; arrowhead). (<b>K</b>, <b>L</b>) Quantification of Pax2a+ cells reveals a significant increase in the number of Pax2a+ cells in the facial (<b>K</b>) and glossopharyngeal and vagal placodes (<b>L</b>) of the transplanted sides versus contralateral sides (Wilcoxon matched-pairs signed rank test: **P<0.01; error bars: standard error of mean; n=8 embryos). Abbreviations: f, facial placode; g+v glossopharyngeal/vagal placode; ov, otic vesicle. Scale bars: 50 µm (A, I); 25 µm (G).</p

Model of Jip3's role in retrograde transport of lysosomes and pJNK.

<p>Our data support a model in which Jip3 (green star) serves as a necessary adapter for retrograde transport of pJNK (yellow hexagon) and lysosomes (red oval). This interaction serves to attach these cargoes to the dynein motor complex and, in the case of lysosomes, likely requires interaction with dynein light intermediate chain (DLIC). Global retrograde transport initiation is unaffected with loss of Jip3 as dynein heavy chain, dynactin and other dynein cargos (late endosomes and autophagosomes) do not accumulate in <i>jip3^nl7</i> mutant axon terminals.</p

Jip3 scaffolds lysosomes to DLIC for retrograde transport.

<p>(A,B) Stills from a wildtype imaging session at 3 dpf in which Lamp1-EGFP (A) and Jip3-mCherry (B) co-transport was analyzed (Video S10). Pink and yellow arrowheads point to two retrograde Jip3/Lamp1 positive cargos. (C,D) Kymographs generated from this imaging session for individual cargos. (E) Schematized kymograph of co-transport. Yellow lines denote Jip3-positive lysosomes moving in the retrograde direction. (F,G) mTangerine-DLIC expression in a wildtype (F) and <i>jip3^nl7</i> mutant (G) NM1 axon terminal at 3 dpf. (H,I) Stills from analysis of Lamp1 (H) and DLIC (I) co-transport at 3 dpf in a wildtype (Video S11). Green arrow-anterograde co-labeled puncta. Yellow arrowhead-DLIC positive lysosome undergoing retrograde transport. (J) The ratio of DLIC positive lysosomes moving in the retrograde direction was significantly decreased in <i>jip3^nl7</i> mutants (ANOVA, *-<i>p</i><0.05; Anterograde-Ant; Retrograde-Ret). (K–M) Kymographs from this imaging session and schematized kymograph depicting co-labeled anterograde lysosomes in green and retrograde in yellow.</p

pJNK levels were elevated in <i>jip3^nl7</i> axon terminals.

<p>(A–H) Immunolabeling for active JNK (pJNK; red in merge; white in single channel) in proximal (NM1) and distal (NM3) neuromasts at 2 and 5 dpf. pJnk levels were elevated in all axon terminals in <i>jip3^nl7</i> mutants (A–I; arrowheads). (I,J) Mean fluorescent intensity (background subtracted; see Materials and Methods for details) of pJNK and total JNK (tJNK) labeling in NM1 axon terminals and the pLL ganglion (pLLg) at 5 dpf. (ANOVA, post-hoc contrasts; *-<i>p</i><0.001). Scale bars = 10 µm.</p

Jip3, an actively transported protein, was necessary for axon extension and the prevention of axon terminal swellings.

<p>(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 <i>neurod:EGFP</i> 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) <i>jip3^nl7</i> mutants displayed truncated pLL nerves and distal pLL nerve thinning (C″) as well as swollen axon terminals in all NMs (NM3 shown in C′). 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 <i>phox2b:EGFP</i> transgenic reporter, were also truncated in <i>jip3^nl7</i> 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 <i>jip3^nl7</i> mutation, which generates a premature stop codon at amino acid 184. (G,H) <i>In situ</i> hybridization analysis revealed that <i>jip3</i> was expressed in the central and peripheral nervous systems at 2 dpf in wildtype but was lost in <i>jip3^nl7</i>. (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 <i>neurod:EGFP</i> 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.</p