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[Preprint]. 2024 Sep 27:2024.09.20.614050. Originally published 2024 Sep 21. [Version 2] doi: 10.1101/2024.09.20.614050

Figure 8. En1 CRISPR loss-of-function causes a loss of limb frequency in juvenile Xenopus laevis.

Figure 8.

A-C. Characterization of En1 CRISPR mutant animals. En1 sgRNA and Cas9 protein were injected at one cell stage to generate bilateral mutant animals, resulting in loss of En1 immunoreactivity from both sides of the spinal cord at NF39–40 (A right, white), or in one cell at two-cell stage to generate unilateral mutant animals with loss of En1 immunoreactivity only from the En1 CRISPR side of the spinal cord at NF54–55 (B right, white). TIDE analysis reveals high efficiency of En1 sgRNA in generating NF44–48 bilateral, and ~25% mutation rate for juvenile unilateral CRISPR animals (C; n = 8 for WT, n = 36 for En1 bilateral mutants, n = 8 for En1 unilateral mutants). Scale bar, 50 μm.

D-H. Range and coordination of movement are unaffected in juvenile unilateral En1 CRISPR mutant animals. WT (D) and unilateral En1 CRISPR (E) juvenile frogs with SLEAP skeleton (yellow) superimposed on animal image. PCA plots represent the position of the fore and hind limb and their range of movement during 256 random frames and show no visible difference in range between WT, uninjected and En1 CRISPR sides (D-E, right; hip and shoulder, yellow; knee and elbow, orange; ankle and wrist, red; foot, brown). Scale bar in D and E indicates the color-code of the first principal component of variation of the aligned fore and hind limb positions. Unilateral En1 CRISPR mutant knees show similar mean angle (F) and angle range (G) as WT. Left-right coordination between knee joints is also unaffected in unilateral En1 CRISPR animals, as their pattern of movement resembles the bilateral synchronicity of WT (H; +1 = bilateral synchronous, 0 = random, −1 = alternate synchronous). n = 13 for WT, n = 8 for unilateral En1 CRISPR.

I-K. Lower dominant frequency in juvenile En1 CRISPR mutant hindlimbs. Mean power spectrum of the knee oscillation shows only one peak in the low frequency bin (0.9–4.5 Hz, dark gray) for WT and unilateral En1 CRISPR animals (I). At the knee joints, the amount of movement, represented by the sum of the power, is similar between WT and unilateral En1 CRISPR animals (J). However, the dominant frequency of the knees is lower in unilateral En1 CRISPR animals compared to WT (K; WT vs En1 ½ CRISPR, p = 0.0002). n = 13 for WT, n = 8 for unilateral En1 CRISPR.