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

Figure 4. Fluorescent in situ hybridization (FISH) reveals vascular signal for both mfsd2aa and mfsd2ab at 5 dpf.

(A) FISH for mfsd2aa (left), mfsd2ab (middle) and mouse Gfap negative control (right) in Tg(kdrl:mCherry) 3 dpf, 5 dpf and adult brain tissue. FISH for mfsd2aa (left) reveals no vascular expression above background at 3 dpf, high levels at 5 dpf and low levels in adult blood vessels. FISH for mfsd2ab (middle) reveals high levels of vascular signal at 3 and 5 dpf and negligible vascular signal in adults. Neither Mfsd2a paralogue was expressed exclusively in the vasculature, outlined in white, at any time examined and mfsd2ab displayed higher overall expression throughout the larval brain than mfsd2aa. Scale bar represents 10 µm. (B) Quantification of the number of transcript puncta per blood vessel area in 3 dpf, 5 dpf and adult brain sections after background (mouse Gfap expression) subtraction. N = 6 sections from at least 3 different fish.

Figure 4—source data 1. Fluorescent in situ hybridization (FISH) and qPCR quantification for mfsd2aa and mfsd2ab.
elife-47326-fig4-data1.xlsx (9.3KB, xlsx)
DOI: 10.7554/eLife.47326.016

Figure 4.

Figure 4—figure supplement 1. FISH reveals consistent vascular slc2a1a signal throughout BBB development.

Figure 4—figure supplement 1.

FISH for slc2a1a (turquoise) and mfsd2aa (green) in Tg(kdrl:mCherry) 3 dpf, 5 dpf and adult brain tissue. FISH for slc2a1a (turquoise) reveals distinct vascular signal at all time points examined, whereas mfsd2aa (green) vascular signal appears at 5 dpf and is maintained at low levels in adults. Scale bar represents 10 µm.
Figure 4—figure supplement 2. Zebrafish have two Mfsd2a paralogues, mfsd2aa and mfsd2ab.

Figure 4—figure supplement 2.

(A) Protein alignment of human MFSD2A, mouse Mfsd2a, zebrafish mfsd2aa, and zebrafish mfsd2ab illustrated with a hydrophobicity scale. Red amino acids are hydrophobic and blue amino acids are hydrophilic. Both zebrafish paralogues are highly similar to the human and mouse proteins. Green boxes highlight the genetic lesions in (B) and the black boxes mark the predicted premature stop codons caused by these mutations in mfsd2aahm37/hm37 and mfsd2abhm38/hm38 mutants. Numbers mark amino acid residues with mutations that have been shown to impact Mfsd2a function:1. Andreone et al. (2017), 2. Guemez-Gamboa et al. (2015), 3. Harel et al. (2018). (B) Sanger sequencing of the mfsd2aahm37 and mfsd2abhm38 mutations. Mfsd2aahm37/37 mutants have a 7 base pair deletion in exon 2 that is predicted to lead to a premature stop codon at amino acid 82 (A; black box). Mfsd2abhm38/hm38 mutants have an 8 base pair insertion (red letters) and a 19 base pair deletion in exon 5 that is predicted to lead to a premature stop codon at amino acid 175 (A; black box). (C) qPCR for mfsd2aa and mfsd2ab mRNA levels in wild-type (black), mfsd2aahm37/hm37 (magenta) and mfsd2abhm38/hm38 (green) mutants at 5 dpf. Mfsd2aa mutants display decreased levels of mfsd2aa and mfsd2ab compared to wild-type fish. Interestingly, mfsd2ab mutants display increased levels of mfsd2aa and decreased levels of mfsd2ab.