FIGURE SUMMARY
Title

The catalytic activity and secretion of zebrafish RNases are essential for their in vivo function in motor neurons and vasculature

Authors
Ferguson, R., Holloway, D.E., Chandrasekhar, A., Acharya, K.R., Subramanian, V.
Source
Full text @ Sci. Rep.
Fig. 4

Effects of NCI-65828 and terrein on zebrafish spinal neurons. (A) Experimental plan for drug treatment of embryos at 10 hpf prior to specification of primary motor neurons and primary motor axon outgrowth. (B) Experimental plan for drug treatment of embryos at 18 hpf after specification of primary motor neurons but prior to primary motor axon outgrowth. (C) Diagrams of embryos at 27 and 36hpf with regions analysed highlighted (D) Islet1 immunostaining at 27 and 36 hpf in the developing spinal cord of embryos treated from 10 hpf with NCI-65828 or terrein. Increased gaps between islet+ nuclei seen (white asterisks). A/P, anterior/posterior. (E) Quantification of motor and Rohon-Beard sensory neurons in the spinal cord at the indicated time-points over the three regions anterior (A) to posterior (P) from 20 embryos (two replicates with 10 embryos each) shows a significant decrease (*p < 0.05) after treatment at 10 hpf. Regions counted and binned as shown by dashed regions in panel C and D.
Fig. 5

Axonal defects in zebrafish embryos treated with NCI-65828 and terrein. Immunostaining for Znp1 in Fli:GFP zebrafish treated with NCI-65828 or terrein from (A) 10 hpf or (B) 18 hpf. Znp1 staining shows defects in the extension and branching of the caudal primary motor neuron (CaP, white dotted lines) extending from the spinal cord (sc). In addition to shortened axons, additional aberrant branching can be seen in treated embryos at positions more proximal to the spinal cord when compared to untreated, which branch only at the distal regions of the axon. Axons in embryos treated at 10 hpf also appear to loop and branch anteriorly at 36 hpf frequently while untreated continue to extend towards the posterior. Treatment from 18 hpf still results in reduced axon length at 27 hpf but to a lesser degree than those treated at 10 hpf. Aberrant branching is still present but with increased branching at the distal tip. By 72 hpf CaP axons appear positioned normally in treated and untreated alike. Observations from 10 embryos in two replicates show significant effects. Detailed quantification can be found in Figure S1. Defects are still seen in the rostral and medial primary motor neurons (RoP and MiP, red and blue dotted lines) and secondary motor neurons (not shown). See Figure S2 for quantification. Scale bars 50 µm.

 
Fig. 6

Vascular defects in zebrafish embryos treated with NCI-65828 and terrein. Immunostaining for GFP in Fli:GFP zebrafish treated with terrein or NCI-65828 from 10hpf (A) shows retarded inter-somitic vessel formation (ISV) at 27 hpf (B) which then recover by 36hpf after NCI-65828 and 20 µM terrein where they span somites from the dorsal aorta (DA) to the dorsal longitudinal anastomotic vessel (DLAV) but not with 30 µM terrein which remain significantly shorter. At 27 hpf over 80% of ISV have not progressed past the midline (C). Immunostaining for GFP in Fli:GFP zebrafish treated with terrein or NCI-65828 from 18 hpf (D) show similar but less severe retardation of ISV growth at 27 hpf and no difference in ISV length at 72 hpf (E). Treatment with NCI-65828 and 30 µM terrein results in less branching of the ISV (F) and 30 µM terrein in particular appears to cause aberrant orientations in the branches (D, 72 hpf dotted line). Scale bars 50 µm. Error bars SEM. *P < 0.05. Quantification made of trunk vessels above the yolk sac extension from 10 embryos in two replicate experiments.
Fig. S1

Quantification of neuronal defects in terrein treated zebrafish

(a) Immunostaining for Znp1 in Fli:GFP zebrafish treated with Terrein or NCI65828 from 10hpf and observed at 27 or 36 hpf. (b) Immunostaining for Znp1 in Fli:GFP zebrafish treated with terrein or NCI65828 from 18 hpf and observed 27 or 72 hpf.  (b) Quantification of primary motor neuron axon length at the indicated time after treatment from 10hpf.  (c) Quantification of primary motor neuron branching at the indicated time after treatment from 10 hpf. (d) Immunostaining for Znp1 in Fli:GFP zebrafish treated with Terrein or NCI-65828 from 18 hpf to 27 or 72 hpf.  (e) Quantification of primary motor neuron axon length at the indicated time after treatment from 18 hpf.  (f) Quantification of primary motor neuron branching at the indicated time after treatment from 18 hpf. Scale bars 50µm. Error bars SEM. * = P<0.05. Quantification made of seven motor axon projections on the left side (1-7, anterior to posterior) above the yolk sac extension from two replicate experiments of ten embryos each.
Fig. S3

Myosin heavy chain immunostaining shows no disruption to underlying body plan after treatment with NCI-65828 or terrein

Immunostaining for Myosin heavy chain in Fli:GFP zebrafish treated with terrein or NCI65828 from 10hpf to 27 or 36 hpf (a) or 18 hpf to 27 or 72 hpf (b). Insets show enlarged regions of main figure with one unit bounded by a dotted line. Scale bars 50µm.
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Sci. Rep.
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