XB-IMG-144369
Xenbase Image ID: 144369
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Figure 4.
Phosphorylation of the JNK site (S189) is essential for the function of hnRNP K in axon outgrowth. Transverse sections of spinal cord through stage 37/38 embryos. Immunostaining (magenta) for N-β-tubulin (A, C, E, G) and NF-M (B, D, F, H) was used to assay rescue of hnRNP K MO phenotype. The midline is represented by a dotted line, and the injected side (+MO) was unilaterally injected at the two-cell stage with hnRNP K MO and mRNA encoding the fluorescent fusion protein indicated (green). A, B, EGFP by itself rescued neither the axonless phenotype (A1, A2) nor the loss of NF-M expression (B1, B2) caused by hnRNP K MO knockdown. NF-M was expressed (*), and motor axons (arrows) were readily visible extending from the spinal cord into somites on the uninjected side, neither of which could be seen on the injected side. C, D, Unilateral coinjection of EGFP–hnRNP K with the hnRNP K MO yielded axons and NF-M expression on both the injected and uninjected sides, indicating the unmutated hnRNP K fusion protein was fully functional in the intact organism. E, F, A serine-to-alanine mutation of the JNK site on hnRNP K (S189A) abrogated the function of hnRNP K in both axon outgrowth and NF-M expression. G, H, A serine-to-aspartic acid mutation (S189D) successfully rescued both axon outgrowth (arrows) and NF-M expression (*). Scale bars, 20 μm. I, J, Reproducibility of phenotypes was assessed using quantitative immunofluorescence to determine rescue of axon outgrowth (I; N-β-tubulin staining in somites) and translation of an mRNA target of hnRNP K (J; NF-M staining in spinal cord). Degrees of rescue and p values are described fully in Results. ns, Nonsignificant; *p < 0.05, one-way ANOVA with Tukey's post hoc test; 5 sections per embryo, n = 3 embryos per condition. Image published in: Hutchins EJ and Szaro BG (2013) Copyright © 2013. This image is reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View |