Figure 2. A novel Mfsd2aD96A mouse abolishes the lipid transport function of Mfsd2a and displays microcephaly. See also Figures S1 and S2; Tables S1 and S2.
(A) Gene targeting strategy for the generation of Mfsd2aD96A mice. Magnified view of the genomic region that contains residue D96 in exon 3 of Mfsd2a. CRISPR/cas9 genome editing introduces the D96A point mutation (red box), as well as two additional silent mutations in codons 97/98 that generate a SpeI restriction site (black underline) for screening purposes. For additional details, see STAR methods. (B–C) Immunostaining at P4 of Mfsd2a protein (green) reveals vascular expression (purple, PECAM) in the neocortex of both Mfsd2a+/+ control (B) and Mfsd2aD96A mutants (C). n = 3 animals per genotype. Scale bar, 100 μm. (D–E) Immunostaining at P4 reveals absence of Mfsd2a protein expression in non-BBB-containing lung vasculature (purple, PECAM) of both Mfsd2a+/+ control (D) and Mfsd2aD96A mutants (E). n = 3 animals per genotype. Scale bar, 100 μm. (F) Western blot from P4 brain lysate of Mfsd2aD96A mutants and littermate Mfsd2a+/+ controls. (G) Quantification shows equal Mfsd2a protein levels between Mfsd2aD96A mutants and littermate Mfsd2a+/+ controls. Tubulin (loading control). n = 3 littermate pairs per genotype. Data are mean ± s.e.m.; n.s., not significant (Student’s t-test). (H) The lipid profile of Mfsd2aD96A mutant brains closely mimics that of Mfsd2a−/− mutant brains at P4. Dot plot representation of Mfsd2a−/−/Mfsd2a+/+ log-ratio (x-axis), compared to Mfsd2aD96A/Mfsd2a+/+ log-ratio (y-axis) of phospholipid levels measured by mass spectrometry. Each dot represents a single lipids species; red line: y=x; R2 = 0.898. Mfsd2aD96A experiment: n = 6 animals per genotype; Mfsd2a−/− experiment: n = 4 animals per genotype. (I) Mfsd2aD96A mutants exhibit microcephaly. Quantification of brain weight at P4. n = 5 animals per genotype. Data are mean ± s.e.m.; ** P < 0.01 (Student’s t-test).