FIGURE SUMMARY
Title

The transcription factor Jun is necessary for optic nerve regeneration in larval zebrafish

Authors
Sarich, S.C., Sreevidya, V.S., Udvadia, A.J., Svoboda, K.R., Gutzman, J.H.
Source
Full text @ PLoS One

Jun expression in the developing retina is downregulated by 48 hpf.

Whole mount in situ hybridization of jun expression in Tg(isl2b:GFP) embryos and larvae from 24 hpf – 120 hpf. (A-C) jun expression in the anterior region of the embryo at 24, 36, and 48 hpf, dorsal views. A. Arrowhead indicates jun expression in the optic lobe. B. Arrowhead indicates jun mRNA staining in the retinal progenitor cell layer. C.jun expression is absent in the ganglion cell layer, arrowhead. (A’-C’) jun sense controls in the anterior region. (D-I’) jun expression and sense controls in lateral views of dissected eyes from 24 hpf - 120 hpf. D.jun expression is present throughout the optic cup. A dotted line outlines the lens mass (Lm) and arrowhead indicates the most concentrated jun expression surrounding the lens mass. E. The retinal progenitor cell layer displays jun expression, outlined. This expression, arrowhead, cups the lens. (F-I) jun expression is undetectable in the ganglion cell layer at 48 hpf- 120 hpf. Arrowheads indicate the ganglion cell layer of the now formed retina. Asterisks indicate the outer pigmented epithelial layer. (A-C) Anterior is toward the top, A-B, dorsal view, C, ventral view. A,D, n = 44; A’,D’ n = 6; B,E, n = 47; B’,E’, n = 5; C,F, n = 34; C’,F’, n = 4; G, n = 23; G’, n = 8; H, n = 26; H’, n = 4; I, n = 25; I’, n = 7. Lm =  lens mass; L =  lens. Scale bars =  100 µm.

Larval zebrafish optic system, optic nerve transection, and axon regeneration timeline.

A. Diagram of the uninjured 5 dpf zebrafish larvae optic system, dorsal view, anterior to the bottom. B. Diagrams and images of Tg(isl2b:GFP) larvae demonstrating the visual system, axonal injury, and regeneration process from 0 hpt - 72 hpt. Red arrows indicate points of optic nerve injury in the left eye. L, lens; ot, optic tectum; on, optic nerve. Scale bar =  100 µm.

Jun mRNA expression is induced early upon optic nerve transection and RGC axon regeneration.

(A–B) Labels describing stage for each dissected eye used for in situ hybridization experiments. (C, C’) In situ hybridization of jun in uninjured eyes at 5 dpf zebrafish. (D–I) In situ hybridization of jun in uninjured, right eye controls of larval zebrafish during optic nerve regeneration at each time point indicated. (D’–I’) jun expression in injured, left eyes during optic nerve regeneration at each time point indicated. (C–I’) Arrowheads indicate ganglion cell layer. 5 dpf uninjured n = 18; 6 hpt, n = 40; 24 hpt, n = 33; 48 hpt, n = 24; 72 hpt, n = 35; 96 hpt, n = 49;120 hpt, n = 49. J. RT-qPCR quantification of jun expression during optic nerve regeneration in the retina. The fold changes of jun during three separate replicates are plotted as dotted lines (green, blue, and purple), the average of the three replicates is plotted is shown as the thick, solid gray line. One-way ANOVA with Tukey’s Multiple Comparison post-test was used to evaluate all pairwise comparisons between time points and determine significance. As a result, fold change at 24 hpt (age: 6 dpf) was statistically significant compared to all other timepoints. 0 hpt, 6 hpt, 72 hpt, 96 hpt, 120 hpt, P < 0.001; 48 hpt, P < 0.01. J. Each time point for each replicate represents n = 30 pooled dissected retinas. (K–L) Diagrams of larvae showing post transection time points next to whole head in situ hybridization for jun comparing uninjured versus injury-induced jun expression in the same animal. White arrow indicates the site of transection. Arrowheads indicate ganglion cell layer. L, lens; ot, optic tectum;. Scale bar =  100 µm.

Early induction of DN-Jun results in developmental defects associated with jun expression.

A. Graphical timeline of experimental workflow. Each DN-Jun strain (mke14Tg, mke15Tg, and mke17Tg) was crossed with Tg(isl2b:GFP); and progeny were heat shocked as described. Fish that were positive for the DN-Jun protein are referred to as DN-Jun(+), siblings that did not express DN-Jun are referred to as DN-Jun(-). (B–C’) Representative brightfield images following experimental procedure outlined in A. B.Tg(isl2b:GFP) X Tg(mke17Tg) DN-Jun(-) fish, 72 hpf, lateral view. C. Tg(isl2b:GFP) X Tg(mke17Tg) DN-Jun(+) fish, 72 hpf, lateral view. B’. Lower jaw and ceratobranchial arches outlined in green as region of interest, and yellow dotted line showing jaw extension in DN-Jun(-) fish. C’. Red outline of lower jaw and ceratobranchial arches as region of interest and yellow dotted line marking jaw extension past the central point of the eye in DN-Jun(+) fish. m =  Meckel’s cartilage, pq =  palatoquadrate cartilage, cb =  ceratobranchial arches. Scale bar =  100 µm.

Induction of DN-Jun inhibits endogenous jun expression during larval optic nerve regeneration.

A. Experimental workflow and RT-qPCR results for evaluation of jun expression during optic nerve regeneration in Tg(isl2b:GFP) fish. At 5 dpf, transections were performed on the left optic nerve. Retinas were dissected and RNA was extracted at 0 hpt, 6 hpt, 24 hpt, 48 hpt, and 72 hpt to use for RT-qPCR. B. Experimental workflow and RT-qPCR results for evaluation of jun expression during optic nerve regeneration in Tg(isl2b:GFP) x Tg(mke15Tg) DN-Jun(+) progeny. Larvae were heat shocked at 4 and 5 dpf, after which they were screened, and transections were performed on the left optic nerve. Retinas were dissected and RNA was extracted at 0 hpt, 6 hpt, 24 hpt, 48 hpt, and 72 hpt to use for RT-qPCR. Each time point for each replicate represents n =  30 pooled retinas. Two-way ANOVA with Bonferroni Multiple Comparisons was performed to determine statistical significance. ** =  p < 0.01; **** =  p < 0.0001.

Induction of DN-Jun diminishes capacity for optic nerve regeneration.

A. Experimental workflow. Left optic nerves of Tg(isl2b:GFP) X Tg(mke15Tg) progeny were transected after the second heat shock at 5 dpf then allowed to regenerate through 72 hpt (age: 8 dpf) when they were live imaged and analyzed. (B–E) Representative live fluorescent images of 72 hpt (age: 8 dpf) DN-Jun expressing larvae and the four potential regeneration outcomes after optic nerve transection. Red asterisks =  site of injury. B. Primarily contralateral axon regeneration, visible GFP labeled axons navigate to the opposite side’s optic tectum, white arrow. C. Contralateral and ipsilateral axon regrowth were both visible, and it was not possible to determine a dominant path, white arrows. D. Visible regenerating axons were primarily directed ipsilaterally, white arrow. E. No visible regeneration occurred; a nerve stump was present, white arrow. F. 100% stacked bar graph of regeneration progress in DN-Jun(-) and DN-Jun(+) fish. Color code indicates the primary axon growth trajectory identified. Green =  contralateral axon growth. Yellow =  both contralateral and ipsilateral axon growth. Orange =  ipsilateral axon growth. Red =  nerve stump, with no visible axon growth. Chi-square analysis was used to determine the significance between DN-Jun(-) and DN-Jun(+) regeneration phenotypes. The nerve stump phenotype was significantly increased in DN-Jun(+), ** P =  0.0025. Graph represents five biological replicates, DN-Jun(-) (negative for DN-Jun after heat shock) n =  73; DN-Jun(+) (positive for DN-Jun after heat shock) n =  87. ot =  optic tectum, L =  lens. Scale bar =  100 µm.

Putative Jun targets display varied expression patterns during larval optic nerve regeneration in control and Jun knockdown conditions.

(A–H) RT-qPCR of jun and putative Jun target genes showing average expression during regeneration in Tg(isl2b:GFP) progeny (green lines) and Tg(isl2b:GFP) X Tg(mke15Tg) DN-Jun(+), Jun knockdown, progeny (orange lines). I: Heatmap showing difference in average fold change of jun and its putative targets’ expression between Tg(isl2b:GFP) and DN-Jun(+) conditions. A–H. Average fold change of three individual replicates were plotted for each condition and timepoint. Each timepoint required n =  90 retinas (30 retinas per replicate for 3 replicates) for both Tg(isl2b:GFP) and DN-Jun(+) experiments. Student’s T-Test was used to determine statistical significance between control and knockdown conditions for each timepoint. *  =  p < 0.05, ** =  p < 0.01, *** =  p < 0.001, **** =  p < 0.0001. All transcripts and primers for these data are shown in Table 1 and transcript ID can be found in S2 Table.

Acknowledgments
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