Abstract
Purpose:
The photoreceptor outer segment is an elaborate primary cilium specialized for photon detection. Anchoring of the basal body at the apical membrane is a prerequisite for outer segment extension, but little is known about the processes that regulate this event. We hypothesized that the microtubule motor cytoplasmic dynein 1 is required for basal body localization prior to photoreceptor ciliogenesis. Cannonball mutant zebrafish carry a nonsense mutation in the heavy chain subunit of cytoplasmic dynein 1 (dync1h1), and the mikre oko mutant has a nonsense mutation in the p150Glued subunit of Dynactin (dctn1a), a key Dynein1 regulatory complex. Previous studies on these mutants showed inner segment organelle positioning defects as well as impaired outer segment morphogenesis. We utilized these mutants to evaluate the requirement of Dynein1 based motility for proper basal body positioning.
Methods:
Cannonball and mikre oko mutant fish were crossed to the Tg(-5actb2:cetn2-GFP) transgenic line, which expresses a centrin-GFP fusion protein from the beta-actin promoter and labels basal bodies. Basal body positioning relative to the outer limiting membrane was assayed at multiple time points in frozen sections counterstained with phalloidin.
Results:
Both cannonball and mikre oko fish underwent a significant degree of retinal degeneration by 4dpf, with rounded nuclei and disorganized lamination frequently observed. The apical actin network was disorganized and often absent in mutant fish, but GFP+ basal bodies were present in areas with an intact outer limiting membrane. Where basal bodies were observed, they were positioned at the expected distance from the OLM.
Conclusions:
Basal bodies in cannonball and mikre oko fish localize properly to the apical membrane in those areas of retina that establish an apical actin network. The patches of retina lacking phalloidin reactivity have no observable basal bodies and may represent areas that lose cell polarity after exhaustion of the maternal protein.
Keywords: 698 retinal development