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. 2022 Oct 12;11:e74270. doi: 10.7554/eLife.74270

Figure 2. klc4uw314 mutants have reduced Rohon-Beard peripheral axon branching.

(A–B) Stills taken at approximately 24 hpf from long term light sheet movies of wild type (A) or klc4uw314 mutant (B) embryo development Scale bar = 150µm. (C) Diagram of a 24 hpf embryo indicating the area over the yolk tube extension imaged for axon analysis, with the standard region used for analysis outlined by the white dashed rectangle. (D–E) Representative examples of HNK-1 stained embryos showing peripheral axon branching in wild type (D) and klc4uw314 I embryos. (F–G) Quantification of axon branching across three technical replicates in wild type (n=32) and klc4uw314 mutant (n=32) embryos (F) and DMSO (n=12) and kinesore (n=17) treated embryos (G). Error bars = SEM. Statistical significance was measured by comparing the area under the curve for (F–G) using t-test. For (F), *p=0.0378 for entire graph. For (G), *p=0.0484 for shaded region. (H–I) Representative examples of HNK-1 stained embryos showing peripheral axon branching in DMSO (H) and kinesore treated (I) embryos. Scale bars for D,E,H,I = 50 µm.

Figure 2—source data 1. The number of branches crossing a selected region of interest (1-8) for wild type and klc4uw314 mutant embryos.
Figure 2—source data 2. The number of branches crossing a selected region of interest (1-8) for wild type embryos treated with either 2% DMSO or 100 µM Kinesore.

Figure 2.

Figure 2—figure supplement 1. Schematic describing our branch analysis method.

Figure 2—figure supplement 1.

A standard sized ROI (ROItrunk) from the trunk over the yolk tube extension is selected with the top of the ROItrunk aligned to the bottom of the central axon fascicle. Eight 3 µm ROIs (white and green, ROIn, n=1–8) are selected at regularly spaced intervals along ROItrunk. Each ROIn is duplicated and then thresholded into a binary image. This binary image is run through Particle Analysis in ImageJ, which counts the number of particles present with gating for size and shape characteristics. We use the number of particles counted to represent the number of branches passing through ROIn.
Figure 2—video 1. A wild type Tg(–3.1ngn1:gfp-caax) embryo imaged by light sheet microscopy over 11.6 hr to show sensory axon development, from approximately 18 to 29.6 hpf.
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Anterior is to the left.
Figure 2—video 2. A klc4uw314 mutant Tg(–3.1ngn1:gfp-caax) embryo imaged by light sheet microscopy over 8.6 hr to show sensory axon development, from approximately 19 to 27.6 hpf.
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Anterior is to the left.