Inactivating mutations in MFSD2A, required for omega-3 fatty acid transport in brain, cause a lethal microcephaly syndrome

Abstract

Docosahexanoic acid (DHA) is the most abundant omega-3 fatty acid in brain, and, although it is considered essential, deficiency has not been linked to disease1,2. Despite the large mass of DHA in phospholipids, the brain does not synthesize it. DHA is imported across the blood-brain barrier (BBB) through the major facilitator superfamily domain–containing 2a (MFSD2A) protein3. MFSD2A transports DHA as well as other fatty acids in the form of lysophosphatidylcholine (LPC). We identify two families displaying MFSD2A mutations in conserved residues. Affected individuals exhibited a lethal microcephaly syndrome linked to inadequate uptake of LPC lipids. The MFSD2A mutations impaired transport activity in a cell-based assay. Moreover, when expressed in mfsd2aa-morphant zebrafish, mutants failed to rescue microcephaly, BBB breakdown and lethality. Our results establish a link between transport of DHA and LPCs by MFSD2A and human brain growth and function, presenting the first evidence of monogenic disease related to transport of DHA in humans.

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Figure 1: MFSD2A mutations cause severe microcephaly and ventriculomegaly.
Figure 2: Thr159Met and Ser166Leu MFSD2A mutants display impaired LPC transport.
Figure 3: Zebrafish mfsd2aa morphants show disrupted BBB integrity, lethality and microcephaly.
Figure 4: Total plasma LPC levels and levels of individual LPC species by lipidomic mass spectrometry.

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References

  1. 1

    Svennerholm, L. Distribution and fatty acid composition of phosphoglycerides in normal human brain. J. Lipid Res. 9, 570–579 (1968).

    CAS  PubMed  Google Scholar 

  2. 2

    Söderberg, M., Edlund, C., Kristensson, K. & Dallner, G. Fatty acid composition of brain phospholipids in aging and in Alzheimer's disease. Lipids 26, 421–425 (1991).

    Article  Google Scholar 

  3. 3

    Nguyen, L.N. et al. Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid. Nature 509, 503–506 (2014).

    CAS  Article  Google Scholar 

  4. 4

    Engle, P.L. et al. Strategies to avoid the loss of developmental potential in more than 200 million children in the developing world. Lancet 369, 229–242 (2007).

    Article  Google Scholar 

  5. 5

    Ben-Zvi, A. et al. Mfsd2a is critical for the formation and function of the blood-brain barrier. Nature 509, 507–511 (2014).

    CAS  Article  Google Scholar 

  6. 6

    Daneman, R. et al. The mouse blood-brain barrier transcriptome: a new resource for understanding the development and function of brain endothelial cells. PLoS ONE 5, e13741 (2010).

    Article  Google Scholar 

  7. 7

    Berger, J.H., Charron, M.J. & Silver, D.L. Major facilitator superfamily domain–containing protein 2a (MFSD2A) has roles in body growth, motor function, and lipid metabolism. PLoS ONE 7, e50629 (2012).

    CAS  Article  Google Scholar 

  8. 8

    Nakanishi, H. et al. Cloning and characterization of mouse lung-type acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1). Expression in alveolar type II cells and possible involvement in surfactant production. J. Biol. Chem. 281, 20140–20147 (2006).

    CAS  Article  Google Scholar 

  9. 9

    Ethayathulla, A.S. et al. Structure-based mechanism for Na+/melibiose symport by MelB. Nat. Commun. 5, 3009 (2014).

    Article  Google Scholar 

  10. 10

    Cordat, E., Leblanc, G. & Mus-Veteau, I. Evidence for a role of helix IV in connecting cation- and sugar-binding sites of Escherichia coli melibiose permease. Biochemistry 39, 4493–4499 (2000).

    CAS  Article  Google Scholar 

  11. 11

    Fleming, A., Diekmann, H. & Goldsmith, P. Functional characterization of the maturation of the blood-brain barrier in larval zebrafish. PLoS ONE 8, e77548 (2013).

    CAS  Article  Google Scholar 

  12. 12

    Kok, F.O. et al. Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish. Dev. Cell 32, 97–108 (2015).

    CAS  Article  Google Scholar 

  13. 13

    Chi, N.C. et al. Foxn4 directly regulates tbx2b expression and atrioventricular canal formation. Genes Dev. 22, 734–739 (2008).

    CAS  Article  Google Scholar 

  14. 14

    He, C., Qu, X., Cui, L., Wang, J. & Kang, J.X. Improved spatial learning performance of fat-1 mice is associated with enhanced neurogenesis and neuritogenesis by docosahexaenoic acid. Proc. Natl. Acad. Sci. USA 106, 11370–11375 (2009).

    CAS  Article  Google Scholar 

  15. 15

    Kawakita, E., Hashimoto, M. & Shido, O. Docosahexaenoic acid promotes neurogenesis in vitro and in vivo. Neuroscience 139, 991–997 (2006).

    CAS  Article  Google Scholar 

  16. 16

    Daneman, R., Zhou, L., Kebede, A.A. & Barres, B.A. Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature 468, 562–566 (2010).

    CAS  Article  Google Scholar 

  17. 17

    Bell, R.D. et al. Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging. Neuron 68, 409–427 (2010).

    CAS  Article  Google Scholar 

  18. 18

    Armulik, A. et al. Pericytes regulate the blood-brain barrier. Nature 468, 557–561 (2010).

    CAS  Article  Google Scholar 

  19. 19

    Harata, N. & Iwasaki, Y. The blood-brain barrier and selective vulnerability in experimental thiamine-deficiency encephalopathy in the mouse. Metab. Brain Dis. 11, 55–69 (1996).

    CAS  Article  Google Scholar 

  20. 20

    DePristo, M.A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011).

    CAS  Article  Google Scholar 

  21. 21

    Fromer, M. et al. Discovery and statistical genotyping of copy-number variation from whole-exome sequencing depth. Am. J. Hum. Genet. 91, 597–607 (2012).

    CAS  Article  Google Scholar 

  22. 22

    Tennessen, J.A. et al. Evolution and functional impact of rare coding variation from deep sequencing of human exomes. Science 337, 64–69 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Shui, G. et al. Comparative plasma lipidome between human and cynomolgus monkey: are plasma polar lipids good biomarkers for diabetic monkeys? PLoS ONE 6, e19731 (2011).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the US National Institutes of Health, including P01HD070494 (to J.G.G. and N.C.C.), R01NS048453 (to J.G.G.), K99NS089943 (to A.G.-G.), and U54HG003067 to the Broad Institute and U54HG006504 to the Yale Center for Mendelian Disorders (M.G.), grant CBRG/069/2014 from the Singapore Ministry of Health's National Medical Research Council (to D.L.S.), Singapore National Research Foundation Competitive Research Program grant 2007-04 (to M.R.W.) and the National University of Singapore's Life Sciences Institute (to M.R.W.). Sequencing was provided in part by a gift from BGI to Rady Children's Hospital, San Diego for undiagnosed patients. Human brain samples were provided by S. Roy (University of California, San Diego).

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M.S.Z., M.K., T.B.-O., K.K.V. and R.O.R. recruited subjects and analyzed the clinical data. E.S., J.S. and B.C. interpreted exome results. J.G.G. and D.L.S. conceived and designed the project. N.A. prepared human brain samples. M.G. and S.G. provided sequencing. A.G.-G. performed genetic analysis to identify MFSD2A mutations. L.N.N. performed lipid transport studies, lipidomics, thin-layer chromatography (TLC) and confocal microscopy. H.Y. and N.C.C. performed zebrafish morpholino studies, and A.G.-G. analyzed and interpreted the data. B.R., D.Q.Y.Q., B.H.W. and B.C.T. assisted with cloning, immunoblots and imaging. A.C.-G. and M.R.W. provided expertise in mass spectrometry. D.L.S., L.N.N., A.G.-G. and J.G.G. wrote the manuscript.

Corresponding authors

Correspondence to David L Silver or Joseph G Gleeson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Homozygosity map of the affected individual from family 1422.

Homozygosity plot showing homozygous blocks (red) in the affected individual from family 1422, with homozygous MFSD2A mutation. Gray, homozygous block comprising MFSD2A overlaps in all affected individuals. Arrow, location of MFSD2A.

Supplementary Figure 2 Homozygosity map of the affected individual from family 1825.

Homozygosity plot showing homozygous blocks (red) in the affected individual from family 1825, with homozygous MFSD2A mutation. Gray, homozygous block comprising MFSD2A overlaps in all affected individuals. Arrow, location of MFSD2A.

Supplementary Figure 3 Chromatograms from Sanger sequencing.

Chromatograms from Sanger sequencing of the fathers (heterozygous) and affected individuals (homozygous) from families 1422 and 1825 and of an unaffected sibling or non-related control (reference normal homozygous) showing the mutations (arrows).

Supplementary Figure 4 MFSD2A is expressed in the endothelial cells of microvessels in human fetal brain.

MFSD2A (red) is highly expressed in endothelium and colocalizes with the glucose transporter GLUT1 (green) in human fetal brain. Arrows show endothelial cells in blood brain vessels. Scale bar, 20 μm.

Supplementary Figure 5 Expression of MFSD2A in human tissues.

RT-PCR (top) and qPCR (bottom) across human adult tissues shows expression in all tissues tested except for skeletal muscle and heart. GAPDH was used as a loading control.

Supplementary Figure 6 Biological incorporation of radiolabeled LPC-[14C]oleate into phosphatidylcholine (PC).

Cells expressing human MFSD2A (WT) and mutants p.Thr159Met (p.T159M) and p.Ser166Leu (p.S166L) or empty plasmid (mock) were incubated with LPC-[14C]oleate for 1 h. Lipids were extracted and analyzed using TLC for phospholipids. PC, phosphatidylcholine; LPC, lysophosphatidylcholine.

Supplementary Figure 7 mfsd2aa and mfsd2ab are expressed in the nervous system of zebrafish embryos.

Whole-mount embryo in situ hybridization with mfsd2aa and mfsd2ab riboprobes at 24, 48 and 96 h.p.f. Both mfsd2aa and mfsd2ab were detected in the nervous system of zebrafish embryos at all stages examined. Whole-mount embryo in situ hybridization with sense mfsd2aa (negative control) and huc (neuronal marker; positive control) riboprobes is shown at 96 h.p.f.

Supplementary Figure 8 Transport activity of zebrafish Mfsd2aa and Mfsd2ab.

(a) Transport of 100 µM LPC-[14C]DHA, LPC-[14C]oleate or LPC-[3H]palmitate after 30 min in cells overexpressing zebrafish Mfsd2aa and Mfsd2ab and human MFSD2A proteins in HEK293 cells. (b) Biological incorporation of radiolabeled LPC-[14C]oleate into phosphatidylcholine (PC). Cells expressing Mfsd2aa and Mfsd2ab or transfected with empty plasmid were incubated with 100 µM LPC-[14C]oleate for 1 h. Lipids were extracted and analyzed using TLC for phospholipids. (c) Quantification of the PC and LPC bands from the TLC plate shown in b. PC, phosphatidylcholine; LPC, lysophosphatidylcholine. Experiments were performed twice in triplicate. Data are expressed as means ± s.e.m. ***P < 0.001.

Supplementary Figure 9 henotypes of mfsd2aa and control morphants.

Injection of zebrafish embryos with mfsd2aa morpholino (MO) caused lethality (~80%) at 24 h.p.f. Representative lateral and dorsal images are shown for surviving control and mfsd2aa morphants at 24 and 96 h.p.f. Arrow, cardiac edema; asterisk, hydrocephalus.

Supplementary Figure 10 Injection with 2,000-kDa dextran.

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected and control embryos at 3 d.p.f. mfsd2aa MO (1 ng) was coinjected with wild-type zebrafish mfsd2aa mRNA (50 ng), wild-type human MFSD2A mRNA (50 ng), or p.Thr159Met (p.T159M) or p.Ser166Leu (p.S166L) mutant human MFSD2A mRNA (50 ng). Colocalization of dextran (green) and cranial blood vessels (red) is shown. See Supplementary Videos 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24.

Supplementary Figure 11 Injection with 10-kDa dextran.

Intracardiac injection of 10-kDa dextran into mfsd2aa morpholino (MO)–injected and control embryos at 3 d.p.f. Dorsal and lateral views are from 2 min after injection. mfsd2aa MO (1 ng) was coinjected with wild-type zebrafish mfsd2aa mRNA (50 ng), wild-type human MFSD2A mRNA (50 ng), or p.Thr159Met (p.T159M) or p.Ser166Leu (p.S166L) mutant human MFSD2A mRNA (50 ng). Colocalization of dextran (green) and cranial blood vessels (red) is shown. See Supplementary Videos 25,26,27,28,29,30,31,32,33,34,35,36.

Supplementary Figure 12 mfsd2aa morphants exhibit brain hemorrhage.

Injection of mfsd2aa morpholino (MO) in zebrafish embryos caused brain hemorrhage (10%) at 3 d.p.f. Representative images of control and mfsd2aa morphants with brain hemorrhage (arrowhead) are shown.

Supplementary Figure 13 Axial T2 MRI images.

Axial T2 MRI images showing the absence of evidence for blood-derived products as determined by this level of resolution in affected children.

Supplementary Figure 14 mfsd2aa morpholino.

Location of the binding site for the mfsd2aa translation-blocking morpholino (mfsd2aa MO) relative to the human and zebrafish mRNAs. Red, start codon; black, morpholino-binding sequence; grey, adjacent sequence.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13, Supplementary Tables 1–6 and Supplementary Note. (PDF 2004 kb)

Supplementary Video 1

Intracardiac injection of 2,000-kDa dextran into control embryos at 3 days post-fertilization (d.p.f.). Side view 0 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to brain parenchyma. (MOV 1799 kb)

Supplementary Video 2

Intracardiac injection of 2,000-kDa dextran into control embryos at 3 days post-fertilization (d.p.f.). Dorsal view 0 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to brain parenchyma. (MOV 1462 kb)

Supplementary Video 3

Intracardiac injection of 2,000-kDa dextran into control embryos at 3 days post-fertilization (d.p.f.). Side view 40 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to brain parenchyma. (MOV 1764 kb)

Supplementary Video 4

Intracardiac injection of 2,000-kDa dextran into control embryos at 3 days post-fertilization (d.p.f.). Dorsal view 40 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to brain parenchyma. (MOV 1397 kb)

Supplementary Video 5

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (1 ng) (MO)–injected embryos at 3 days post-fertilization (d.p.f.). Side view 0 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1768 kb)

Supplementary Video 6

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (1 ng) (MO)–injected embryos at 3 days post-fertilization (d.p.f.). Dorsal view 0 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1649 kb)

Supplementary Video 7

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (1 ng) (MO)–injected embryos at 3 days post-fertilization (d.p.f.). Side view 40 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1471 kb)

Supplementary Video 8

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (1 ng) (MO)–injected embryos at 3 days post-fertilization (d.p.f.). Dorsal view 40 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1810 kb)

Supplementary Video 9

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with zebrafish wild-type MFSD2A mRNA (50 ng). Side view 0 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to the absence of dextran extravasation into the brain parenchyma. (MOV 1759 kb)

Supplementary Video 10

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with zebrafish wild-type MFSD2A mRNA (50 ng). Dorsal view 0 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to the absence of dextran extravasation into the brain parenchyma. (MOV 1351 kb)

Supplementary Video 11

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with zebrafish wild-type MFSD2A mRNA (50 ng). Side view 40 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to the absence of dextran extravasation into the brain parenchyma. (MOV 1796 kb)

Supplementary Video 12

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with zebrafish wild-type MFSD2A mRNA (50 ng). Dorsal view 40 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to the absence of dextran extravasation into the brain parenchyma. (MOV 1651 kb)

Supplementary Video 13

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with human wild-type mfsd2aa mRNA (50 ng). Side view 0 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to the absence of dextran extravasation into the brain parenchyma. (MOV 1531 kb)

Supplementary Video 14

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with human wild-type mfsd2aa mRNA (50 ng). Dorsal view 0 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to the absence of dextran extravasation into the brain parenchyma. (MOV 1699 kb)

Supplementary Video 15

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with human wild-type mfsd2aa mRNA (50 ng). Side view 40 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to the absence of dextran extravasation into the brain parenchyma. (MOV 1712 kb)

Supplementary Video 16

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with human wild-type mfsd2aa mRNA (50 ng). Dorsal view 40 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to the absence of dextran extravasation into the brain parenchyma. (MOV 1490 kb)

Supplementary Video 17

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with mutated p.Ser166Leu (p.S166L) human MFSD2A mRNA (50 ng). Side view 0 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1524 kb)

Supplementary Video 18

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with mutated p.Ser166Leu (p.S166L) human MFSD2A mRNA (50 ng). Dorsal view 0 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1383 kb)

Supplementary Video 19

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with mutated p.Ser166Leu (p.S166L) human MFSD2A mRNA (50 ng). Side view 40 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1297 kb)

Supplementary Video 20

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with mutated p.Ser166Leu (p.S166L) human MFSD2A mRNA (50 ng). Dorsal view 40 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1333 kb)

Supplementary Video 21

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with mutated p.Thr159Met (p.T159M) human MFSD2A mRNA (50 ng). Side view 0 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1305 kb)

Supplementary Video 22

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with mutated p.Thr159Met (p.T159M) human MFSD2A mRNA (50 ng). Dorsal view 0 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1718 kb)

Supplementary Video 23

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with mutated p.Thr159Met (p.T159M) human MFSD2A mRNA (50 ng). Side view 40 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1288 kb)

Supplementary Video 24

Intracardiac injection of 2,000-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with mutated p.Thr159Met (p.T159M) human MFSD2A mRNA (50 ng). Dorsal view 40 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1877 kb)

Supplementary Video 25

Intracardiac injection of 10-kDa dextran into control embryos at 3 days post-fertilization (d.p.f.). Side view 2 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to brain parenchyma. (MOV 1754 kb)

Supplementary Video 26

Intracardiac injection of 10-kDa dextran into control embryos at 3 days post-fertilization (d.p.f.). Dorsal view 2 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to brain parenchyma. (MOV 1811 kb)

Supplementary Video 27

Intracardiac injection of 10-kDa dextran into mfsd2aa morpholino (1 ng) (MO)–injected embryos at 3 days post-fertilization (d.p.f.). Side view 2 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1370 kb)

Supplementary Video 28

Intracardiac injection of 10-kDa dextran into mfsd2aa morpholino (1 ng) (MO)–injected embryos at 3 days post-fertilization (d.p.f.). Dorsal view 2 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1479 kb)

Supplementary Video 29

Intracardiac injection of 10-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with human wild-type MFSD2A mRNA (50 ng). Side view 2 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to the absence of dextran extravasation into the brain parenchyma. (MOV 1483 kb)

Supplementary Video 30

Intracardiac injection of 10-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with human wild-type MFSD2A mRNA (50 ng). Dorsal view 2 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to the absence of dextran extravasation into the brain parenchyma. (MOV 1259 kb)

Supplementary Video 31

Intracardiac injection of 10-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with zebrafish wild-type mfsd2aa mRNA (50 ng). Side view 2 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to the absence of dextran extravasation into the brain parenchyma. (MOV 1549 kb)

Supplementary Video 32

Intracardiac injection of 10-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with zebrafish wild-type mfsd2aa mRNA (50 ng). Dorsal view 2 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to the absence of dextran extravasation into the brain parenchyma. (MOV 1288 kb)

Supplementary Video 33

Intracardiac injection of 10-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with mutated p.Ser166Leu (p.S166L) human MFSD2A mRNA (50 ng). Side view 2 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1444 kb)

Supplementary Video 34

Intracardiac injection of 10-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with mutated p.Ser166Leu (p.S166L) human MFSD2A mRNA (50 ng). Dorsal view 2 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1577 kb)

Supplementary Video 35

Intracardiac injection of 10-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with mutated p.Thr159Met (p.T159M) human MFSD2A mRNA (50 ng). Side view 2 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1739 kb)

Supplementary Video 36

Intracardiac injection of 10-kDa dextran into mfsd2aa morpholino (MO)–injected embryos at 3 days post-fertilization (d.p.f.). mfsd2aa MO (1 ng) was coinjected with mutated p.Thr159Met (p.T159M) human MFSD2A mRNA (50 ng). Dorsal view 2 min after dextran injection. Colocalization of dextran (green) and cranial blood vessels (red). Arrow pointing to dextran extravasation into the brain parenchyma. (MOV 1608 kb)

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Guemez-Gamboa, A., Nguyen, L., Yang, H. et al. Inactivating mutations in MFSD2A, required for omega-3 fatty acid transport in brain, cause a lethal microcephaly syndrome. Nat Genet 47, 809–813 (2015). https://doi.org/10.1038/ng.3311

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