Fig. 5

Smaller Kupffer’s vesicles (KV) in MZcarmil3 mutants are composed of fewer cells and have reduced number of cilia. (A) Confocal images of embryos at the 10-14 somite stage stained to visualize cilia and the KV, with antibodies to acetylated tubulin (Ac-tubulin, Green in Merge), and to atypical PKC (aPKC, Red in Merge) respectively. With anti-aPKC staining, the KV lumen appears as a relatively dark area, owing to the absence of cells inside the vesicle. WT and two different carmil3 alleles are shown. The merge panel shows examples of how the KV lumen was outlined for calculation of area; the outline is based on both Ac-tubulin and aPKC images. Panels B through D are graphs of parameters quantified from the images, with the color scheme for WT and mutants as indicated. In each panel, the plotted values are the mean, and the error bars correspond to one standard deviation. (B) Area of the KV. Horizontal dotted line corresponds to the value for area of KV found to be necessary for robust L/R patterning by Gokey and colleagues (Gokey et al., 2016). Values for the two mutants differ from the value for WT based on Student’s t-test (p<0.005 (actual=0.0006 and 0.0005)). (C) Number of cilia per KV. Horizontal dotted line corresponds to the value for number of functional cilia per KV (30) found to be necessary for robust left / right patterning by Sampaio and colleagues (Sampaio et al., 2014). Values for the two mutants differ from the value for wild type based on Student’s t-test (p<0.005 (Actual=0.0001 and 0.0004)). (D) Length of cilia. Values for the two mutant lines do not differ from the value for WT embryos based on Student’s t-test (p=0.3277 and >0.9999). Values of N (embryos counted) as follows: WT, 9; MZcarmil3^stl413, 8; MZcarmil3^sa19830, 4 for panels B to D. (E) Panel E shows a second experimental series with a different set of animals than those in panels A-D. Left side: The number of cells in the KV at the 6-somite stage is reduced in MZcarmil^stl413 (29.4) relative to WT (32.5), without statistical significance. Each was injected with a control RNA (~12.5 pg of Histone2B-RFP). Right side: The total area of KV is proportional to the number of cells. The same KVs examined for the cell number on the left were measured for area on the right.