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Angela Gauthier, John Borchert, Anna Larson, Janey L. Wiggs; Abnormal ocular development and reduced vision in zebrafish after morpholino-mediated knockdown of ARHGAP33. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3428.
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Early-onset glaucoma has a strong genetic factor, but only 20% of affected patients have mutations in known disease-causing genes. Whole exome sequencing of 27 early-onset glaucoma families found 3 families with ARHGAP33 mutations, suggesting ARHGAP33 as a promising candidate. This study explores the functional impact of ARHGAP33 mutations by using morpholino-mediated knockdown of the ARHGAP33 ortholog in zebrafish embryos.
Embryos at the 1-8 cell stage were injected with 2-3 nL of 0.4mM ARHGAP33 translation-blocking (TB), 0.4 mM ARHGAP33 splice-site blocking (SS), or 0.5 mM standard (STD) control morpholinos, and one group was uninjected (UI). Eye area, head area, and body length were measured at 3 days post fertilization (dpf). Vision was studied at 5 dpf by visual motor response (VMR), a device that measures activity levels in response to light change.
Each group included approximately 100 embryos, and survival at 5 dpf was 37% for TB, 45% for SS, 53% for STD, and 84% for UI. STD and UI had no differences in eye area (66,481 ± 4,245 mm2 and 66,942 ± 4,067 mm2, p = 0.65), eye/head area (0.25 ± 0.02 and 0.25 ± 0.01, p = 0.49), and body length (3,408 ± 116 mm and 3,458 ± 100 mm, p = 0.08). STD and SS also had no differences in these variables (66,041 ± 4,245 mm2, p = 0.76; 0.25 ± 0.02, p = 0.87; 3,355 ± 120 mm, p = 0.15). 50% of TB fish had a severe phenotype of significantly smaller eye area (31,903 ± 7,686 mm2, p < 0.001), eye/head area (0.19 ± 0.04, p < 0.001), and body length (1,858 ± 295 mm, p < 0.001) than STD. The remaining TB fish also had a significantly smaller eye area (57,568 ± 2,415 mm2, p < 0.001) and body length (2,768 ± 424 mm, p = 0.04) than STD but had no difference in eye/head area (0.26 ± 0.02, p = 0.19).VMR testing of 24 fish from each group showed no difference between the SS, STD, and UI fish. Severe phenotype TB fish had no baseline movement due to skeletal dysmorphology. When these were excluded, the TB fish still did not show a significant activity spike in response to light, unlike the other groups.
The ARHGAP33 TB morpholino had a significant negative effect on zebrafish eye and body development and vision. ARHGAP33 knockout mice have been shown to express less TRKB, which is needed for retinal ganglion cell survival. Together, these findings suggest a possible role for ARHGAP33 in glaucoma-related neurodegeneration.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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