Figure 5-figure supplement 2.

Figure 5-figure supplement 2.

(A) Representative maximum intensity projection images of the midbrain of 5 dpf uninjected controls, mfsd2aa and mfsd2ab crispant fish. Crispant fish were injected with Cas9 protein and two unique guide RNAs from those used to generate mutants to target either mfsd2aa or mfsd2ab. Mosaic mfsd2aa crispants display increased DBP-EGFP tracer permeability at similar levels to mfsd2aa^hm37/hm37 mutants (Figure 5) and displayed an 80% reduction in mfsd2aa levels by qPCR. Conversely, mfsd2ab crispants do not appear to have any BBB permeability defects, as observed in mfsd2ab^hm38/hm38 mutants (Figure 5), while displaying a 60% reduction in mfsd2ab levels by qPCR. The scale bar represents 50 µm. (B) Quantification of DBP-EGFP tracer intensity in the midbrain and hindbrain parenchyma of uninjected controls (black), mfsd2aa crispants (magenta) and mfsd2ab crispants (green). DBP-EGFP intensity was measured in the midbrain and averaged to generate a single value per fish that was normalized to circulating tracer levels. Each individual fish measured is displayed as a single point. The mean and the standard error are drawn in black. ****p<0.0001 by two-way ANOVA.