Article Figures & Data
Figures
- Figure 1.
Generation of mutations in the zebrafish kiss1 gene using CRISPR/Cas9. A, B, Dorsal view of zebrafish larvae, labeled with an antibody to Kisspeptin1 (A; cyan) and the Kisspeptin receptor (B; yellow). These fish are from the Et(SqKR11) transgenic line (Lee et al., 2010) and express red fluorescence in afferents from the entopeduncular nucleus (basal ganglia) that terminate in the neuropil of the ventral habenula. The green arrowhead indicates the habenula. The speckles appear to be nonspecific label. C, A schematic of the kiss1 gene. There are two introns. The signal sequence is shown in blue, and the region containing the active Kisspeptin1 peptide is shown in green. D, Partial sequence of the kiss1 gene. The signal sequence is indicated by the blue bar. The position of the two guide RNAs are indicated by the black bars. E, The sequence of the three mutant alleles, together with predicted translations. The asterisks indicate stop codons. The black bars indicate inserted sequences. F, A kiss1sq1sj−/− fish, following labeling with the Kisspeptin1 antibody. No signal could be detected in the habenula. Scale bar = 25 µm. Pa, pallium; rHb, right habenula; lHb, left habenula. Images are single optical sections, with anterior to the left.
- Figure 2.
The behavior of kiss1 mutants in an active avoidance assay. A, Schematic of the two-way active avoidance chamber. B, Number of crossovers before shock delivery for mutants (n = 31) and WT siblings (n = 37). The black symbols are average values. C, Number of crossovers in a group of mutants (n = 22) trained over two consecutive days. D, Response to the first shock, as measured by swim speed in the first second immediately after the shock. E, Mean swim speed during the 20-min habituation period. F, Response to the first exposure to the red light. This was measured by the ratio of the mean speed in the 2 s after light onset to the mean speed in the 5 s before light onset. In all cases error bars indicate 95% confidence interval.
- Figure 3.
The effect of Kisspeptin1 on vHb neurons. A, Representative trace of cellular response (top, black) in a cell to voltage steps (bottom, gray). These recordings were done in the presence of 1 μm tetrodotoxin to block network activity. B, Difference current obtained after bath application of 5 μm K-10 (n = 10 cells). The same protocol as in A was done before and after bath application of K-10 for each cell. The traces obtained after were subtracted from the traces obtained before application of the peptide for the same cell. C, Dosage response of the difference current. Superimposed scatter and box plots for the difference currents induced at 25 mV for four different K-10 concentrations (10 nm, 100 nm, 1 μm, and 5 μm). Difference currents induced at 10 and 100 nm are significantly different from that induced at 5 μm (*p < 0.05, Kruskal–Wallis nonparametric test followed by post hoc Tukey–Kramer method of multiple comparison, n = 10 cells for each K10 dose).
- Figure 4.
The effect of shock on raphe neurons in kiss1 mutants and siblings. A, Change in fluorescence of GCaMP6f in neurons in the superior raphe of kiss1sq1sj mutants (blue trace) and wild-type siblings (red trace). 50 V was applied for 1 s, 15 s after the start of imaging, at the point indicated by the arrow. The traces indicate mean value while the shading indicates SEM. B, Average z-score of cells in the 15-s period after onset of shock. Each circle represents a single cell, and cells are arranged according to fish. The black dot indicates mean, and the bars indicate 95% confidence interval.
Tables
Figure Data structure Type of test Result 2B Normal distribution Shapiro–Wilk test W = 0.969; p = 0.0929 t test p = 0.0024 Effect size (Cohen’s d) d = 0.77 2C Nonnormal distribution Shapiro–Wilk test W = 0.889; p = 0.001 Wilcoxon matched pairs test p = 0.011 Effect size (adjusted Cohen’s d) d = 0.91 2D Normal distribution Shapiro–Wilk test W = 0.966; p = 0.057 t test p = 0.13 2E Normal distribution Shapiro–Wilk test W = 0.986; p = 0.673 t test p = 0.22 2F Nonnormal distribution Shapiro–Wilk test W = 0.829; p = 0.001 Mann–Whitney U test p = 0.98 3C Nonnormal distribution Kruskal–Wallis nonparametric test followed by post hoc Tukey–Kramer method of multiple comparison p = 0.012; p = 0.029 (10 nm vs. 5 µm); p = 0.034 (100 nm vs. 5 µm) 4B Multilevel model analysis Intracluster correlation 0.46 Chi-squared (df = 1) 5.5971 p = 0.015
In this issue
