One hypothesis for the transient nature of EGFP/XKlp3B-ST transgene expression in
Xenopus rod photoreceptors is that disruption of kinesin II function within the cell by the dominant negative-acting transgene eventually causes cellular apoptosis and thus loss of transgene-expressing rods. To test this hypothesis, we examined 0.5-μm sections of wild-type and transgenic retinas from 7- to 9-day-old tadpoles containing the EGFP/XKlp3B-ST transgene, the EGFP/XKlp3B-ST and the XKlp3A-ST transgenes, or the EGFP transgene alone. Shown in
Figure 2 are examples of control and transgenic retinal sections that were analyzed to compare the numbers of rods and cones. Note that in the section from the EGFP/XKlp3B-ST transgenic tadpole (
Fig. 2A , between filled arrows) there was an area where there were fewer rods than cones compared with other areas in the same retina and with the retinas from an EGFP transgenic
(Fig. 2C) or a wild-type
(Fig. 2D) tadpole of the same age (areas between open arrows). The reduction in the number of rods was even more pronounced in transgenic retinas that contain both dominant negative-acting kinesin II subunit transgenes. In the double transgenic tadpole shown in
Fig. 2B (between the filled arrows), there were very few remaining rod photoreceptors, whereas there were a significant number of intact cones. There was little evidence of cone degeneration or degeneration of cells in any of the other retinal layers, suggesting that the effect is specific only for the rods expressing the dominant negative-acting transgenes.
The effect of kinesin II dominant–negative expression on the retinal rods was quantified by analyzing the rod-cone ratio of transgenic kinesin II retinas and comparing the average ratios with those of control retina. The average rod-cone ratios were determined by counting the numbers of rods and cones from three to five retinal sections from three to eight tadpoles fixed 7, 8, and 9 days after fertilization. Shown in
Figure 3 are the combined data from tadpoles with retinas that were fixed 7 to 9 days after fertilization. Tadpoles expressing the EGFP/XKlp3B-ST had an average rod-cone ratio of 1.1 ± 0.019 (
n = 21 tadpole retinas). The average ratio from the transgenic tadpoles containing both kinesin II subunit dominant negative proteins was even lower, 0.68 ± 0.13 (
n = 13 tadpole retinas), whereas the ratios from transgenics containing EGFP alone (1.7 ± 0.095,
n = 10 tadpole retinas) or from wild-type (1.6 ± 0.052,
n = 18 tadpole retinas) were higher. The lower rod-cone ratios between the single or double kinesin II transgenic retinas and both EGFP and/or wild-type controls are statistically significant (
P < 0.001). In addition, a statistical difference (
P < 0.005) was found in the ratios between the retinas containing EGFP/Klp3B-ST alone and EGFP/XKlp3B-ST and XKlp3A-ST together, suggesting that expression of both dominant negative-acting subunits had a greater effect on reducing the rod-cone ratio than expression of only one subunit. The rod-cone ratio data from tadpoles fixed 7, 8, and 9 days after fertilization were combined, because no significant differences in the rod-cone ratios were detected between retinas fixed at 7, 8, or 9 days in either single or double kinesin II transgenic or control retinas. In addition, no significant difference was found between the data from control EGFP and wild-type retinas of any age, supporting evidence from previous studies that no deleterious effects on rod survival are produced by EGFP transgene expression. The presence of the transgene was confirmed by PCR of genomic DNA for all the tadpoles containing kinesin II transgenes.
The dramatic effects of kinesin II dominant negative-acting transgene expression in the
Xenopus tadpole retina are evident in one animal containing both EGFP/XKlp3B-ST and XKlp3A-ST transgenes, which was examined at the ultrastructural level
(Fig. 4A) . Very few rods were detected, whereas the cones (indicated by an asterisk in the cone lipid droplet) occurred at regular intervals in the photoreceptor layer. The control tadpole retina expressing the EGFP transgene alone
(Fig. 4B) , similar to the tadpole retina containing both dominant negative-acting kinesin II subunits, is from a stage-48 animal that was fixed 7 days after fertilization. Unlike the kinesin II transgenic animal, the retina expressing only EGFP contained more rods than cones, and the rods had developed long, orderly outer segments. In the one rod (
Fig. 4A , arrow) found in the kinesin II transgenic retinal section, little of the outer segment remained, and what remained contained disordered disks. In addition, the inner segment of the kinesin II transgenic rod contained vacuoles and vesicles containing dense material that were not seen in the control EGFP rods. The nucleus of the kinesin II transgenic rod was apoptotic and highly condensed compared with the cone nuclei in the outer nuclear layer and with the nuclei in the inner nuclear layer below. Although the cones and the other layers of the retina in the kinesin II transgenic animal shown in
Figure 4A looked relatively normal, retinas from a few kinesin II transgenic animals also displayed degeneration in cones and other retinal cell layers as well as rods that may reflect apoptotic effects caused by the degeneration of transgenic rods in later stages of tadpoles or in tadpoles containing a greater number of rods expressing the kinesin II transgene or containing a greater level of transgene expression.