FIGURE SUMMARY
Title

Biological variability hampers the use of skeletal staining methods in zebrafish embryo developmental toxicity assays

Authors
Hoyberghs, J., Ball, J., Trznadel, M., Beekhuijzen, M., Burbank, M., Wilhelmi, P., Muriana, A., Powles-Glover, N., Letamendia, A., Van Cruchten, S.
Source
Full text @ Reprod. Toxicol.
Fig. 1

Zebrafish larvae at 5 dpf with bone (red) and cartilage (blue) structures that were stained with the alizarin red- alcian blue staining. Left panel (A) shows a ventral view. Right panel (B) shows a lateral view. Each structure (except for uot and cot) is indicated in only one of the orientations, namely in the orientation where it was scored. Uot and cot were scored by looking at both positions. The abbreviations are depicted in Table 2 and Table 3. The structures vb* and en* are not present in these images.

Fig. 2

Zebrafish larvae at 5 dpf with bone structures that were stained with the alizarin red live staining. Left panel (A) shows a ventral view. Right panel (B) shows a lateral view. Each structure is indicated in only one of the orientations, namely in the orientation where it was scored. The abbreviations are depicted in Table 2.

Fig. 3

Zebrafish larvae at 9 dpf with bone structures that were stained with the alizarin red live staining. Left panel (A) shows a ventral view. Right panel (B) shows a lateral view. Each structure is indicated in only one of the orientations, namely in the orientation where it was scored. The abbreviations are depicted in Table 2. The left cleithrum (c*) is hidden behind the right cleithrum in this image.

Fig. 4

Mean angle sizes between ceratohyal cartilages in 5 dpf larvae treated with compound 5. All groups were compared to the solvent control group. No significant differences were observed between the 3 test groups or the medium control group and the solvent control group.

Fig. 5

Mean intensity score of fixed AR-stained bones of 5 dpf larvae treated with proprietary compound 5. A) shows the results of replicate 1 and B) shows the results of replicate 2. All groups were compared to the solvent control group. Significant differences between the solvent control and any of the other groups are indicated. P ≤ 0.05 (*) and p ≤ 0.01 (**).

Fig. 6

Mean intensity score of fixed AB-stained cartilages of 5 dpf larvae treated with proprietary compound 5. A) shows the results of replicate 1 and B) shows the results of replicate 2. All groups were compared to the solvent control group. Significant differences between the solvent control and any of the other groups are indicated. P ≤ 0.05 (*) and p ≤ 0.01 (**).

Fig. 8

Mean intensity score of fixed AR-stained bones of 5 dpf larvae treated with proprietary compound 9. A) shows the results of replicate 1 and B) shows the results of replicate 2. All groups were compared to the solvent control group. Significant differences between the solvent control and any of the other groups are indicated. P ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***). The color of the asterisks indicate that the solvent control was significantly more (black) or less (green) intense stained than the other group. The green underlined asterisks indicate that the solvent control was significantly less stained than the medium control.

Fig. 9

Mean intensity score of fixed AB-stained cartilages of 5 dpf larvae treated with proprietary compound 9. A) shows the results of replicate 1 and B) shows the results of replicate 2. All groups were compared to the solvent control group. Significant differences between the solvent control and any of the other groups are indicated. P ≤ 0.05 (*) and p ≤ 0.01 (**).

Fig. 10

Mean angle sizes between ceratohyal cartilages in 5 dpf larvae treated with compound A. All groups were compared to the solvent control group. Significant differences between the solvent control and any of the other groups are indicated. P ≤ 0.001 (***).

Fig. 11

Mean intensity score of fixed AR-stained bones of 5 dpf larvae treated with proprietary compound A. A) shows the results of replicate 1 and B) shows the results of replicate 2. All groups were compared to the solvent control group. Significant differences between the solvent control and any of the other groups are indicated. P ≤ 0.05 (*). The black asterisks indicate that the solvent control was significantly more intense stained than the other group. The red asterisks indicate that the solvent control was significantly more stained than the medium control

Fig. 12

Mean intensity score of fixed AB-stained cartilages of 5 dpf larvae treated with proprietary compound A. A) shows the results of replicate 1 and B) shows the results of replicate 2. All groups were compared to the solvent control group. No significant differences were observed between the 3 test groups or the medium control group and the solvent control group.

Fig. 13

Mean angle sizes between ceratohyal cartilages in 5 dpf larvae treated with levetiracetam. All groups were compared to the solvent control group. Significant differences between the solvent control and any of the other groups are indicated. P ≤ 0.05 (*).

Fig. 14

Mean intensity score of fixed AR-stained bones of 5 dpf larvae treated with levetiracetam. A) shows the results of replicate 1 and B) shows the results of replicate 2. All groups were compared to the solvent control group. Significant differences between the solvent control and any of the other groups are indicated. P ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.0001 (****). The color of the asterisks indicate that the solvent control was significantly more (black) or less (green) intense stained than the other group.

Fig. 15

Mean intensity score of fixed AB-stained cartilages of 5 dpf larvae treated with levetiracetam. A) shows the results of replicate 1 and B) shows the results of replicate 2. All groups were compared to the solvent control group. Significant differences between the solvent control and any of the other groups are indicated. P ≤ 0.05 (*).

Fig. 16

Calcein stained larval zebrafish at 5 and 9 dpf. Panel A shows the detail of bone formation detectable by calcein staining. Panel B demonstrates the variability between the same spawning group of individuals treated at the same time. Abbreviation: days post-fertilization (dpf).

Fig. 17

Images of AR live stained bones of 5 dpf larvae treated with A) the solvent control, B) 100 µM of compound 5 and C) 1,000 µM of compound 5. The parasphenoid, which is normal in A and B, and malformed in C, is indicated with a white box. All pictures are from larvae of the first replicate and show the larvae in dorsoventral position.

Fig. 18

Mean intensity score of AR live stained bones of 5 dpf larvae treated with proprietary compound 5. A) shows the results of replicate 1 and B) shows the results of replicate 2. Both groups were compared to the solvent control group. Significant differences between the solvent control and the test groups are indicated. P ≤ 0.05 (*) and p ≤ 0.01 (**). The color of the asterisks indicate that the solvent control was significantly more (black) or less (green) intense stained than the other group.

Fig. 19

Mean intensity score of AR live stained bones of 9 dpf larvae treated with proprietary compound 5. A) shows the results of replicate 1 and B) shows the results of replicate 2. Both groups were compared to the solvent control group. Significant differences between the solvent control and the test groups are indicated. P ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***) and p ≤ 0.0001 (****).

Fig. 20

Variability in staining between control larvae of the two replicates of the AR live experiment. A) shows the results using 5 dpf larvae and B) shows the results using 9 dpf larvae. Significant differences between the two groups of solvent controls are indicated. P ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***) and p ≤ 0.0001 (****).

Fig. 21

Variability in staining between two clutches of control larvae (same spawning day). Significant differences between the two clutches are indicated. P ≤ 0.05 (*).

Fig. 22

Variability in staining between subgroups of the same batch of control larvae. In A) the results of batch 1 are shown, in B) the results of batch 2 are shown and in C) the results of another batch (i.e., the solvent control group of the second replicate of the compound 5 experiment at 5 dpf) are shown. For each of these batches, the batch was divided into 3 subgroups of 8 fish. All the subgroups of a batch were compared to each other. Significant differences between the three subgroups are indicated. P ≤ 0.05 (*) and p ≤ 0.01 (**).

Fig. 23

Differences in mean angle size between the medium controls of all fixed staining experiments. P ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***).

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.

Reprinted from Reproductive toxicology (Elmsford, N.Y.), 127, Hoyberghs, J., Ball, J., Trznadel, M., Beekhuijzen, M., Burbank, M., Wilhelmi, P., Muriana, A., Powles-Glover, N., Letamendia, A., Van Cruchten, S., Biological variability hampers the use of skeletal staining methods in zebrafish embryo developmental toxicity assays, 108615, Copyright (2024) with permission from Elsevier. Full text @ Reprod. Toxicol.
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