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. 2019 Aug 20;8:e47326. doi: 10.7554/eLife.47326

Figure 5. Mfsd2aa mutants exhibit increased BBB permeability.

(A) Representative maximum intensity projection images of the brain of wild-type and mfsd2aa mutants injected with a fluorescent 1 kDa NHS tracer (turquoise) at 5 dpf. Mfsd2aa mutants have increased levels of NHS in the midbrain and hindbrain parenchyma outside of the vasculature (magenta; Tg(kdrl:mCherry)) compared to wild-type siblings. (B) Representative maximum intensity projection images of the brain of wild-type and mfsd2aa mutants expressing the fluorescently labeled 80 kDa transgenic serum protein DBP-EGFP (green) at 5 dpf. Mfsd2aa mutants have increased levels of DBP-EGFP in the midbrain and hindbrain parenchyma compared to wild-type siblings. The scale bar represents 50 µm. (C) Quantification of normalized parenchymal tracer intensity in the midbrain and hindbrain of wild-type (black) and mfsd2aa mutants (magenta) reveals that mfsd2aa mutants have significantly increased levels of tracer permeability, both for the injected NHS (A) and the endogenous transgene DBP-EGFP (B). Parenchymal tracer intensity outside of the vasculature was measured and normalized to the blood vessel tracer intensity for both the midbrain and hindbrain in each fish and displayed as a single point. The mean and the standard error are drawn as black lines. ****p<0.0001, ***p<0.001 by t test.

Figure 5—source data 1. Mfsd2aa mutants exhibit increased BBB permeability, mfsd2ab mutants do not.
elife-47326-fig5-data1.xlsx (13.9KB, xlsx)
DOI: 10.7554/eLife.47326.021

Figure 5.

Figure 5—figure supplement 1. Mfsd2ab mutants do not have altered BBB permeability.

Figure 5—figure supplement 1.

(A) Representative maximum intensity projection images of the brain of wild-type and mfsd2ab mutants expressing the fluorescently labeled 80 kDa transgenic serum protein DBP-EGFP (green) at 5 dpf. Mfsd2ab mutants have a similarly low level of DBP-EGFP in the midbrain and hindbrain parenchyma compared to wild-type siblings. The scale bar represents 50 µm. (B) Quantification of normalized parenchymal tracer intensity (NHS and DBP-EGFP) in the midbrain and hindbrain of wild-type (black) and mfsd2ab mutants (green). Mfsd2ab mutants display no significant barrier permeability defects, both for the injected NHS and the endogenous transgene DBP-EGFP (A). Each individual fish measured is displayed as a single point. The mean and the standard error are drawn in black.
Figure 5—figure supplement 2. Mosaic mfsd2aa crispants display increased BBB permeability while mfsd2ab crispants display a wild-type BBB.

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 mfsd2aahm37/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 mfsd2abhm38/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.
Figure 5—figure supplement 3. Mfsd2aahm37/hm37; mfsd2abhm38/hm38 double mutants display similar increased BBB permeability to mfsd2aahm37/hm37 single mutants.

Figure 5—figure supplement 3.

(A–D) Representative maximum intensity projection images of the midbrain of wild-type (A; mfsd2aa+/+; mfsd2ab+/+), mfsd2aahm37/hm37 single mutants (B), mfsd2abhm38/hm38 single mutants (C), and mfsd2aahm37/hm37; mfsd2abhm38/hm38 double mutants (D) expressing the fluorescently labeled 80 kDa transgenic serum protein DBP-EGFP (green) at 5 dpf. Mfsd2aahm37/hm37; mfsd2abhm38/hm38 double mutants display a similar level of increased parenchymal tracer permeability to the mfsd2aahm37/hm37 single mutants. The scale bar represents 50 µm. (E) Quantification of DBP-EGFP tracer intensity in the midbrain parenchyma of wild-type (black), mfsd2aahm37/hm37 mutants (magenta), mfsd2abhm38/hm38 mutants (green), and mfsd2aahm37/hm37; mfsd2abhm38/hm38 double mutants (turquoise). 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.