Figure 1.
Illustration of quantitative measurements of zebrafish body waving.
The fish body (blue curve) is characterized at twenty body locations (red dot). L is the body length, T is the distance between the tail and the center of fish body, is the angular change between two consecutive body locations. Given the coordinates of two consecutive body locations [x(i), y(i)] and [x(i+1), y(i+1)], the angular change is defined as
.
Figure 2.
The body curvature comparison between wild-type zebrafish and transgenic zebrafish.
(A) wild-type zebrafish; (B) transgenic zebrafish; (C) the comparison of curvatures measured along twenty body locations of wild-type zebrafish (green plot) and transgenic zebrafish (red plot), respectively. The fish body (blur curve) is characterized at twenty body locations (red dot) in (A) and (B). In (c), the x-axis represents the fish body location index, and the y-axis represents the body curvature value (in the unit of degree). Note that we extracted the skeleton of the fish body and used twenty control points to describe the posture of fish. These control points are uniformly distributed along the fish body skeleton from the head to the tail. The first point is located at fish head and the last point is located at fish tail. The body curvature is measured at each point location.
Figure 3.
Maps of α-actin:hCLCN1-EGFP and α-actin:hCLCN1-IRES-EGFP constructs used in this study.
(A–C) Three constructs expressing hCLCN1, hCLCN1I553F/H555N, and hCLCN1L844F –green fluorescent protein (EGFP) fusions under the control of the α-actin promoter were used to produce stable transgenic zebrafish lines. EGFP was fused to the C-terminus of the flag-tagged hCLCN1. The Tol2 vector sequences are shown as thick black lines. (D-E) Three constructs expressing hCLCN1, hCLCN1I553F/H555N, hCLCN1L844F under the control of the α-actin promoter were used to produce stable transgenic zebrafish lines. An internal ribosome entry site (IRES) element is inserted in front of EGFP to allow simultaneous expression of hCLCN1 and EGFP protein separately but from the same RNA transcript. The Tol2 vector sequence is shown as thick black lines.
Figure 4.
Characterization of stable transgenic zebrafish embryos using Tg (α-actin:hCLCN1I553F/H555N–EGFP).
(A) A pool of 28 h dpf embryos from the stable transgenic founder fish showing the EGFP expression. Nonfluorescent embryos are nontransgenic siblings. (B) Embryos with positive EGFP expression show the muscle-specific expression in their trunk region. (C) Confocal microscopic image of the trunk of an F1 generation Tg (α-actin:hCLCN1I553F/H555N–EGFP) embryos at 3 dpf. EGFP expression was observed within the zebrafish muscle fibers. (D) Transgenic founders were confirmed by transmission of the CLCN1 transgene to F1 progeny by PCR. Transgenic founders were crossed with WT zebrafish, individual F1 embryos were lysed and PCR were performed using hCLCN1 specific primers and the PCR product analyzed by agarose gel electorphorsis. EGFP-positive embryos are identified based on the presence of the 1.5 kb band (e3, 5, 6, 9 and 10), while the EGFP-negative embryos lack this band (e1, 2, 4, 7, 8, 11 and 12). e; embryo. (E) Transgenic founders were confirmed by Western blot. Protein was extracted from F1 embryos at 2 dpf. Lane 1: WT AB Zebrafish, Lane 2–4: Individual stable transgenic fish expressing the 130 kDa Flag-tagged hCLCN1I553F/H555N–EGFP (see Materials and Methods). Actin was used as a loading control. (F) Transgenic zebrafish embryos at 3 dpf showing low, moderate or high expression of hCLCN1I553F/H555N–EGFP based on GFP intensity. (G) Quantification of hCLCN1I553F/H555N–EGFP expression and classification. For each transgene, 3 siblings of 3 dpf embryos were analyzed. Average pixel intensities (api) for EGFP fluorescence were determined on 5 areas of the trunk and the average for each sibling is plotted. Expression levels of the transgene were arbitrarily classified into low (<10 api), moderate (10–20 api) or high (>20 api). Similar results were obtained for the other transgenes (data not shown).
Figure 5.
The comparison of averaging spatial-temporal body curvature profiles and averaging temporal tail offset profiles.
For each zebrafish, 16-second video sequences at high frame rates were recorded. The video clip was manually split into smaller clips containing one full body waving cycle. The fish body was tracked and the bending degree determined using body curvature and tail offset as criteria. (A–G) averaging spatial-temporal body curvature profiles that are obtained from the following zebrafish: wild-type, hCLCN1-EGFP, hCLCN1L844F-EGFP, hCLCN1I553F/H555N-EGFP, hCLCN1-IRES-EGFP, hCLCN1L844F-IRES-EGFP, hCLCN1I553F/H555N-IRES-EGFP, respectively. We consolidated body curvature measurements calculated at each frame into a matrix. Each column represents the body curvature measurements in one frame. The number of columns is equal to the number of frame of one body waving cycle. We further averaged matrices over all body waving cycles of all zebrafish within same category to obtain an averaging body curvature profile. Red color indicates higher curvature value and larger bending of fish body. The Y-axis represents the body curvature measurements in one frame. The X-axis represents frame index of one video segment of one body waving cycle. (H–I) Consolidated tail offset measurements calculated at each frame into a vector. The vectors are averaged over all body waving cycles of all zebrafish within same category to obtain an averaging tail offset profile. Larger tail offset value indicates smaller bending of fish body.
Figure 6.
Comparison of body curvature, tail offset and swimming distance measurements between wild-type, control and transgenic zebrafish.
We selected the largest body curvature value (A–B); smallest tail offset value (C–D), and travel distance (E–F) from each body waving cycle. All measurements are first averaged over all video clips within same category. The averaged measurements are further divided by the mean measurement of wild-type zebrafish. The measurements are plotted as mean +/− standard deviation. The star symbol in the figure indicates a significant difference (p<0.05) in the Student-t test.