Mrc1a-positive perivascular cells cover the zebrafish brain.

(a) Schematic diagram of an adult zebrafish brain. Dorsal view, rostral at top. Box shows region imaged in panel b. (b) Epifluorescence microscopic image of the optic lobes (top) and cerebellum of the dissected brain of a Tg(mrc1a:eGFP);Tg(kdrl:mCherry) double-transgenic 4 month old adult zebrafish (eGFP and mCherry are shown in green and magenta, respectively). (c,d) Higher magnification confocal images of Kdrl:mCherry-positive blood vessels (magenta) and closely associated Mrc1a:eGFP-positive perivascular cells (green) on the surface of one of the optic lobes of the brain. (e) Confocal image of a transverse section through the brain of a Tg(mrc1a:eGFP) transgenic adult zebrafish, at the level of the optic lobes (top), stained with anti-GFP (green) and DAPI (magenta), n = 2 brains. Box shows region depicted in panel F. (f,g) Higher magnification confocal images of the brain section imaged in panel E, showing Mrc1a:eGFP-positive cells restricted almost exclusively to the surface meningeal layer. Box in f shows region imaged in panel g. Scale bars: 500 µm (b,e), 100 µm (c), 50 µm (d), 20 µm (f,g).

Mrc1a-positive perivascular cells cover the zebrafish brain.

(a,b) Dorsal (a) and ventral (b) confocal images of the dissected brain of a Tg(mrc1a:eGFP);Tg(kdrl:mCherry) double-transgenic adult zebrafish (eGFP and mCherry are shown in green and magenta, respectively). Rostral is to the top. Mrc1a:eGFP cells populate the entire optic tectum on the dorsal and ventral sides but only the ventral side of the forebrain/telencephalon and the dorsal side of the cerebellum. Scale bar: 500 µm.

Mrc1a-positive perivascular cells are present in the developing zebrafish brain, and express lymphatic markers.

(a,b) Confocal images of Mrc1a:eGFP-positive cells (green) and Kdrl:mCherry-positive blood vessels (magenta) on the surface of the brain in 10 dpf (a) and 15 dpf (b) Tg(mrc1a:eGFP);Tg(kdrl:mCherry) double-transgenic zebrafish (n = 3 animals of each age imaged). (c,d) Same images as in panels (a) and (b) with both Kdrl:mCherry blood vessel fluorescence and non-perivascular green fluorescence rendered in grey, Mrc1a-positive perivascular cells are depicted in green. Small arrows in panels a-d are autofluorescent pigment cells on the dorsal head of the zebrafish. Large arrows in panels b and d are facial lymphatics. (e) Epifluorescence microscopic image of Mrc1a:eGFP-positive cells on the optic lobes (left) of the dissected brain of a 1 month-old Tg(mrc1a:eGFP) transgenic zebrafish (n = 3 brains imaged). Panels a-e are dorsal views of the brain surface, with rostral to the left. (f,g) Airyscan confocal images of Mrc1a:eGFP-positive perivascular cells (green) extending along Kdrl:mCherry-positive blood vessels (grey) in 5 dpf Tg(mrc1a:eGFP);Tg(kdrl:mCherry-CAAX) double-transgenic zebrafish (n = 3 animals imaged). (h) Confocal image of Mrc1a:eGFP (green), Lyve1:dsRed (magenta) double-positive cells on the dissected brain of an adult 4 month old Tg(mrc1a:eGFP);Tg(lyve1:dsRed) double-transgenic zebrafish (dorsal view, rostral up), n = 3 brains imaged. (i,j) Higher magnification single-channel confocal images illustrating that the cells on the surface of the brain imaged in panel h are expressing both Mrc1a:eGFP (i) and Lyve1:dsRed (j). (k) Confocal image of Mrc1a:eGFP (green), Prox1a:RFP (magenta) double-positive cells on 4 dpf brain of a Tg(mrc1a:eGFP);Tg(prox1:RFP) double-transgenic animal (lateral view, rostral to the left). l,m, Single channel Mrc1a:eGFP (green; l) and Prox1a:RFP (magenta; m) images of the boxed region in panel k, showing the cells are expressing both transgenes. dpf, days post fertilization. Scale bars: 200 µm (a–d, k), 35 µm (f,g), 500 μm (h), 50 µm (i,j), 100 µm (l,m).

Mrc1a-positive perivascular cells are EdU negative and fail to collect and drain dye like Mrc1a-positive lymphatics.

(a) EdU staining (magenta) of a 5 dpf Tg(mrc1a:eGFP) transgenic embryo where Mrc1a:eGFP-positive cells (green) are not EdU positive (n = 5 animals). Rostral is to the left. (b) Lymphatic vessel (green) on the head of a juvenile Tg(mrc1a:eGFP) transgenic animal showing lymphatic drainage of injected Qdots-705 (blue). (c) FGPs (green) on the brain surface of a Tg(mrc1a:eGFP) transgenic juvenile do not drain injected Qdots-705 (blue). n = 2 injected fish. Scale bars: 100 µm.

Mrc1a-positive perivascular cells are Fluorescent Granular Perithelial cells (FGPs).

(a,b) Yellow/green epifluorescence microscopic images of the optic lobes (top) and a portion of the cerebellum of the dissected brain of a non-transgenic wild type adult zebrafish (n = 3 adult brains). Dorsal views rostral at top. Box in a shows region imaged in panel b, where autofluorescent granules are clearly visible in close association with blood vessels. (c,d) Confocal images of Mrc1a:eGFP-positive perivascular cells (green) filled with numerous vacuoles closely apposed to Kdrl:mCherry-positive blood vessels (grey) on the surface of the optic lobe of a dissected brain of a Tg(mrc1a:eGFP);Tg(kdrl:mCherry) double-transgenic adult zebrafish (n = 3 adult brains). Box in panel (c) shows region imaged in panel d. (e) Confocal and transmitted light (grey) overlay image of Lyve1:dsRed-positive perivascular cells (magenta) filled with large vacuoles (black arrows) in the brain of an adult Tg(lyve1:dsRed) transgenic zebrafish (n = 3 adult brains). Dashed lines demarcate an adjacent blood vessel. (f) Confocal image of the same Lyve1:dsRed-positive cells (magenta) showing that the vacuoles are all yellow autofluorescent (green, white arrows). (g) Confocal image of a transverse section through the brain of an adult mouse, at the level of the cerebral cortex, stained with anti-CD206 (MRC1; green) and DAPI (magenta), n = 3 brains, three sections. (h) Higher magnification confocal images of the brain section imaged in panel g, showing CD206-positive cells on the outermost surface of the brain. (i,j) Confocal images of Mrc1a:eGFP-positive perivascular cells (green) containing numerous vacuoles (red and black) on the surface of an optic lobe of a dissected brain of a Tg(mrc1a:eGFP) transgenic adult zebrafish injected intracranially with India ink. Panel (j) shows transmitted light image. Yellow dotted lines in j represent the outline of Mrc1a:eGFP-positive cells. Black vacuoles contain India ink internalized by Mrc1a:eGFP-positive cells (n = 3/10 injected brains). (k,l) Confocal images of Mrc1a:eGFP-positive perivascular cells (green) on Kdrl:mCherry-positive blood vessels (magenta) on the surface of the optic lobes of dissected brain of a normally (k) fed and high cholesterol diet (HCD) fed (l) Tg(mrc1a:eGFP);Tg(kdrl:mCherry) double-transgenic adult zebrafish (n = 3 per treatment). (m,n) Quantification of average FGP cell length (panel m; T-test, p-value=1.7×10⁻¹⁰, n = 347 FGPs from 3 adult brains) and average vacuole number (panel n; T-test, p-value=9.2×10⁻⁶, n = 354 FGPs from 3 adult brains) in control versus HCD fed Tg(mrc1a:eGFP);Tg(kdrl:mCherry) double-transgenic adult zebrafish. Scale bars: 50 µm (c,d), 10 µm (e,f), 500 μm (g), 20 μm (h), 25 µm (i,j), 50 µm (k,l).

Zebrafish FGPs do not correspond to other known perivascular cell types.

(a) Schematic diagram of cells associated with brain blood vessels, including the vascular endothelial cells, pericytes/smooth muscle cells, radial glia, macrophages/neutrophils, and FGPs. (b) Confocal image of Lyve1:dsRed-positive (magenta) and Pdgfrb:Citrine-positive (green) cells on the dissected brain of an adult Tg(lyve1:dsRed);Tg(pdgfrb:Citrine) double-transgenic zebrafish (n = 2 brains imaged). (c) Higher magnification confocal image of the same field illustrating that the two transgenes are expressed in distinct and separate cell populations - FGPs (large arrows) and pericyte (small arrows). (d) Transmitted light and epifluorescence Mrc1a:eGFP composite image of the dorsal head of a 5 dpf Tg(mrc1a:eGFP) transgenic animal (n = 15 animals). (e) Transmitted light image of the dorsal head of a neutral red-stained 5 dpf Tg(mrc1a:eGFP) transgenic animal (n = 15 animals). Rostral to the left. (f) Confocal image of Lyve1:dsRed-positive (magenta) and Mpx:eGFP-positive (green) cells in the head of a 5 dpf Tg(lyve1:dsRed);Tg(mpx:eGFP) double-transgenic zebrafish (n = 3 animals imaged). Lateral view, rostral to the left. (g) Higher magnification image of the boxed region in panel f, illustrating that the two transgenes are expressed in distinct and separate cell populations - FGP (large arrows) and neutrophils (small arrows). (h–n) Overview image (h) and magnified images of boxed region in panel h at selected time points (i–n) from a time-lapse confocal image series of Lyz:dsRed positive neutrophils (magenta) migrating into and out of the area of a wounded vessel (asterisk in h) on the dorsal brain surface of a 4 dpf Tg(mrc1a:eGFP);Tg(lyz:dsRed) double-transgenic zebrafish (rostral up), (n = 2 animals time-lapse imaged). The migrating neutrophils frequently interact with the FGPs adjacent to the wound site (arrows in panels i,k,m,n). (o) Lateral view confocal image of a 5 dpf Tg(mrc1a:eGFP);Tg(mpeg1:gal4);Tg(UAS:NTR-mCherry) triple-transgenic zebrafish, with Mrc1a:eGFP-positive cells in green and Mpeg1:Gal4/UAS-NTR-mCherry-positive macrophages in magenta. (p), Higher magnification image of the boxed region in panel o (n = 5 animals imaged). (q) Higher magnification image of the boxed region in panel p, showing that the two transgenes are expressed in distinct and separate FGP (white arrows) and macrophage (yellow arrows) cell populations. (r), Lateral view confocal image of the brain (centered on the optic lobes) of a 15 dpf Tg(mrc1a:eGFP);Tg(mpeg1:gal4);Tg(UAS:NTR-mCherry) triple-transgenic zebrafish, with Mrc1a:eGFP-positive cells in green and Mpeg1:Gal4/UAS-NTR-mCherry-positive macrophages in magenta (n = 3 animals imaged). (s), Higher magnification image of the brain in panel r. Yellow box shows magnified image in panel t. (t) Higher magnification image of the boxed region in panel s, showing that the two transgenes are expressed in distinct and separate FGP (white arrows) and macrophage (yellow arrows) cell populations. Rostral is to the left in all images. Scale bars: 100 µm (b, s, t), 50 μm (c, h–n), 200 µm (f,g, o–q).

Radial Glia and FGPs localize to different parts of the brain.

(a) Dorsal view of a Z-projected confocal image generated by the Zebrafish Brain Browser (Marquart et al., 2015) superimposing four 6 dpf Tg(lyve1:dsRed) (green) embryos to Tg(gfap:GFP) (magenta). Rostral is to the left. (b,d) Horizontal views of panel A at the top (b) and bottom (d) of the Z-stack across the entire brain. (c,e) Sagittal views of panel A at the top (c) and bottom (e) Z-sections. White arrows in panels b-e show FGPs. Panels b'-e' show the Gfap:GFP channel only. Panels b'-e' show the Lyve1:dsRed channel only. Scale bar for all panels: 100 μm.

FGPs are not Neural Crest derived.

(a-b) Lateral view of a 3.5 dpf Tg(sox10:dsRed); Tg(mrc1a:eGFP) double transgenic embryo showing mrc1a:eGFP-positive FGPs in green (a) and Sox10:dsRed–positive neural crest derived structures in magenta (a,b; n = 5 animals imaged). (c–f), Dorsal views of the optic lobes of a 5 dpf Tg(sox10:dsRed); Tg(mrc1a:eGFP) double transgenic embryo with mrc1a:eGFP-positive FGPs in green (c,e) and Sox10:dsRed–positive neural crest derived structures in magenta (c-f; n = 5 imaged animals). (e,f) Higher magnification images of the right optic lobe in the yellow box in panel c. Scale bars: 100 μm (a, b), 200 μm (c, d).

Zebrafish FGPs are not derived from definitive hematopoietic progenitors.

(a) Schematic diagram illustrating hematopoietic stem and progenitor cells (HSPCs) differentiation from endothelial cells in the ventral floor of the dorsal aorta (DA). No, Notochord; DA, Dorsal aorta; PCV, Posterior Cardinal Vein. (b,c) Green-to-red photoconversion of the DA in a 24 hpf Tg(egfl7:gal4);Tg(UAS:Kaede);Tg(mrc1a:eGFP) triple-transgenic embryo, showing red/green (b) or red only (c) confocal fluorescence images immediately after photoconversion at 24 hpf (b,c; n = 5 animals photoconverted). (d-h) Confocal images of green (d–g) and red (d,e,f,h) fluorescence in the thymus (d), trunk vessels (e) and dorsal optic tectum FGPs (f–h) in 5 dpf Tg(egfl7:gal4);Tg(UAS:Kaede);Tg(mrc1a:eGFP) triple-transgenic animal subjected to DA photoconversion at 24 hpf. Arrows in panels g and h show Mrc1a:eGFP positive FGPs on the head (g) that are not red fluorescent (h; n = 5/5 photoconverted animals lacking red fluorescent FGPs. Yellow box in notes region shown at higher magnification in panels g and h. (i–l) Confocal imaging of Lyve1:dsRed (magenta; i,k) and Runx1:GFP (green; i–l) fluorescence in the lateral head (i,j) or dorsal head (k,l) of 5 dpf Tg(lyve1:dsRed);Tg(runx1:eGFP) double-transgenic animals (rostral to the left in all panels; n = 3 animals imaged). Large arrows in panels i and j note the Runx1:GFP-positive thymus. Small arrows in panels i-l note Lyve1:dsRed-positive but Runx1:GFP-negative FGPs. (m,n) Confocal imaging of Lyve1:dsRed (magenta) and c-Myb:eGFP (green) fluorescence in the dorsal head of a 5 dpf Tg(lyve1:dsRed);Tg(c-myb:eGFP) double-transgenic animals (rostral to the left in all panels; n = 3 animals imaged). Boxed region in panel m is displayed at higher magnification in panel n. Large arrows note Lyve1:dsRed-positive but c-Myb:eGFP negative FGPs; small arrows note c-Myb:eGFP-positive but Lyve1:dsRed negative hematopoietic cells. Scale bars: 200 μm (b–c, g–k), 100 µm (d), 50 µm (e,f–h,i–n).

FGPs are not derived from primitive hematopoiesis.

(a-d) Dorsal yolk (a,b) and lateral trunk (c, d) transmitted light images of 20 hpf control morpholino (a,c) or pu.1 morpholino (b,d) injected embryos subjected to whole mount in situ hybridization and probed for mpx. Arrows in panels a and c show mpx-positive cells in controls that are absent in pu.1 morpholino-injected animals.( e,f) Lateral view confocal images of 5 dpf Tg(mrc1a:eGFP) transgenic control morpholino (e,g) or pu.1 morpholino (f, h) injected larvae. (g,h) Higher magnification images of brains of the same control morpholino (g) or pu.1 morpholino (h) injected larvae shown in panels e and f, respectively. (i) Quantification of FGP cells present in control vs. pu.1 morpholino-injected animals at 5 dpf (t-test, p-value=0.76). Scale bars: 500 µm (e,f), 200 µm (g,h).

Zebrafish FGPs are not derived from definitive hematopoietic progenitors.

(a) Schematic diagram illustrating hematopoietic stem and progenitor cells (HSPCs) differentiation from endothelial cells in the ventral floor of the dorsal aorta (DA). No, Notochord; DA, Dorsal aorta; PCV, Posterior Cardinal Vein. (b–d) Green-to-red photoconversion of the DA in a 24 hpf Tg(fli:gal4);Tg(UAS:Kaede);Tg(mrc1a:eGFP) triple-transgenic embryo, showing red/green (b,d) or red only (c) confocal fluorescence images either immediately after photoconversion at 24 hpf (b,c) or 12 hr post-photoconversion at 36 hpf (d), (n = 6 animals photoconverted). (e–h) Confocal images of green (e–g) and red (e,f,h) fluorescence in the trunk vessels (e), thymus (f), and dorsal optic tectum FGPs (g,h) in a 5 dpf Tg(fli:gal4);Tg(UAS:Kaede);Tg(mrc1a:eGFP) triple-transgenic animal that had been subjected to DA photoconversion at 24 hpf. Arrows in panels e and f show readily apparent residual red fluorescence in the DA (e) and in the thymus (f). Arrows in panels g and h show Mrc1a:eGFP positive FGPs on the head (g) that are not red fluorescent (h), (n = 6/6 photoconverted animals lack red fluorescent FGPs).

Inhibiting HSPCs specification does not affect FGP formation.

(a,a’,b,b’) Lateral views of 5 dpf Tg(lyve1:dsRed);Tg(mpx:eGFP) double transgenic animals showing the lymphatic vessels and FGPs (magenta) and macrophages (green punctate) in an uninjected (a) or runx1 Fluorescein-tagged morpholino injected (b) animal. Panels a’ and b’ show only the green fluorescence channel. (c,d) Higher magnification lateral views of the boxed regions in panels a and b, respectively, showing Lyve1:dsRed-positive FGPs localized to the optic tectum at 5 dpf. (e,f) Quantification of Mpx:eGFP-positive cells (e, n = 4 animals imaged and quantitated) and Lyve1:dsRed-positive FGPs (f, n = 8 animals imaged and quantitated) in 5 dpf Tg(lyve1:dsRed);Tg(mpx:eGFP) double transgenic animals after runx1 morpholino injection. Scale bars in all panels: 200 µm.

Global analysis of gene expression in FACS-sorted adult zebrafish FGPs.

(a) Confocal microscopic image of the optic lobes (top) and cerebellum of the dissected brain of a Tg(mrc1a:eGFP);Tg(kdrl:mCherry) double-transgenic adult zebrafish (EGFP and mCherry are shown in green and magenta, respectively), n = 10 adult brains. (b) FACS sorting of FGPs (GFP positive) and blood endothelial cells (RFP positive) from Tg(mrc1a:eGFP);Tg(kdrl:mCherry) adult tectal meninges; GFP vs. RFP (mCherry) plot showing gates used for cell collection. (c–f) Plots showing relative expression levels of selected genes in FACS sorted FGPs (EGFP-positive, n=~1000 cells per replicate from a total of ~8000 cells sorted) compared to whole fish. (c) Blood endothelial (left) and lymphatic endothelial (right) markers and factors; (d) macrophage, neutrophils and HSPC markers; e) glial and neurotrophic factors; (f) pericyte markers. Relative expression is plotted on a log two scale. Scale bars: 500 μm (a).

Zebrafish FGPs emerge from the endothelium of the choroidal vascular plexus.

(a), Dorsal (top) and lateral (bottom) view schematic diagrams of approximately 2.5 dpf zebrafish heads with some of their associated vasculature, especially major venous tracts. CVP, choroidal (optic choroidal) vascular plexus; PMBC, primordial midbrain channel; PHBC, primordial hindbrain channel; BA, basilar artery. Adapted from Figure 5B in the Vascular Anatomy of Zebrafish Atlas (Isogai et al., 2001) – see this reference for additional details. (b–e) Confocal images of Mrc1a:eGFP-positive FGPs (green) and Kdrl:mCherry-positive blood vessels (grey) on the surface of the brain in 3 dpf (b,c) and 4 dpf (d,e) Tg(mrc1a:eGFP);Tg(kdrl:mCherry) double-transgenic zebrafish. b and d are lateral views, c and e are dorsal views of the head with rostral to the left. Some residual blood vessel GFP fluorescence was deleted for clarity; see Figure 7—figure supplement 1 4a–d for the original unmanipulated green/magenta images. (f–j) Confocal time lapse imaging of Mrc1a:eGFP-positive (green) FGPs migrating dorsally then medially along Kdrl:mCherry-positive (magenta) blood vessels on the surface of the brain in a 2.5–3 day old Tg(mrc1a:eGFP);Tg(kdrl:mCherry) double-transgenic zebrafish. Panel f shows an overview dorsal image from the 780’ minute time point (h). Panels g-j show magnified views of the boxed region in panel f at 0, 296, 780, and 1416 min time points. (k–n) Dorso-lateral view of a 2.5 dpf Tg(egfl7:gal4);Tg(UAS:Kaede);Tg(mrc1a:eGFP) triple-transgenic embryo readily after photoconverting the OCVP (k, white box). Red dots in k represent eye autofluorescent pigment. (l-n), Higher magnification of the photoconverted OCVP depicted in k (white box) showing expression of red photoconverted Kaede (l,m) and GFP (l,n). (o) Photoconversion on the OCVP results in red Kaede Mrc1a:eGFP-positive cells. Lateral view of a 5 dpf Tg(egfl7:gal4); Tg(UAS:Kaede); Tg(mrc1a:eGFP) triple-transgenic embryo (o) showing Red Kaede, Mrc1a-GFP-positive FGPs (white arrows) on the optic tectum (n = 8/12 embryos showed Red Kaede, with 1.25 ± 1.14 S.D. red FGPs per tectal neuropile).( p–r), Higher magnification views of the boxed region in panel o showing Mrc1a:eGFP (green, panels p and r) and photoconverted Kaede (red, panels p and q) double positive FGPs (white arrows). Rostral is to the left in all panels. Scale bars = 100 µm.

Emerging FGPs on the 3 and 4 dpf zebrafish brain.

(a-d) Confocal images of Mrc1a:eGFP-positive FGPs (green) and Kdrl:mCherry-positive blood vessels (magenta) on the surface of the brain in 3 dpf (a,b) and 4 dpf (c,d) Tg(mrc1a:eGFP);Tg(kdrl:mCherry) double-transgenic zebrafish. Panels a and c are lateral views, panels b and d are dorsal views of the head. Images are the unmanipulated versions of the images shown in Figure 7a–d, where some residual blood vessel GFP fluorescence was deleted for clarity. Rostral is to the left in all panels. Scale bars = 100 µm. Rostral is to the left. Scale bar = 200 μm.

Zebrafish FGPs emerge from the endothelium of the choroidal vascular plexus.

(a) Dorsal (top) and lateral (bottom) view schematic diagrams of approximately 2.5 dpf zebrafish heads with some of their associated vasculature, especially major venous tracts. OCVP, choroidal (optic choroidal) vascular plexus; PMBC, primordial midbrain channel; PHBC, primordial hindbrain channel; BA, basilar artery. Adapted from Figure 5B, The Vascular Anatomy of Zebrafish Atlas (Isogai et al., 2001) – see this reference for additional details. (b) Photoconversion on the OCVP results in red Kaede Mrc1a:eGFP-positive cells. Dorsal view of a 5 dpf Tg(fli:gal4);Tg(UAS:Kaede);Tg(mrc1a:eGFP) triple-transgenic (b) showing FGPs on the optic tectum (box), (n = 9/19 embryos showed Red Kaede, with 1.37 ± 1.98 S.D. red FGPs per tectal neuropile). (c–e) Higher magnification views of the box in panel b showing Mrc1a:eGFP (green) and photoconverted Kaede (red) double positive FGPs (white arrows). Green fluorescence is shown in panels c and d, red fluorescence is shown in panels c and e. Rostral is to the left in all panels. Scale bars: 200 μm (b); 50 μm (b’–d).

Zebrafish FGPs differentiate from the CVP endothelium.

(a) Model diagram showing the location of the CVP imaged during timelapse acquisition in a dorsal (top) and lateral (bottom) orientation.( b–g) Stills from time-lapse movies of cells emerging from the CVP at around 2.5–3 dpf in a Tg(mrc1a:eGFP);Tg(kdrl:mCherry) double transgenic animal. Mrc1a:eGFP vessels are depicted in gray for ease of visualization, Kdrl:mCherry vessels are magenta and emerging FGPs are green (yellow arrows and numbers 1,2). Mrc1a:eGFP-positive FGPs emerge as single cells and stay in contact with the vessels in the perivascular space. (h), Model diagram showing the location of the CVP imaged during timelapse acquisition in a dorsal (top) and lateral (bottom) orientation.( i–k) Emergence of FGPs from the CVP in Tg(mrc1a:eGFP);Tg(kdrl:nlsmCherry) double transgenic animal. Newly emerging FGPs (white, yellow arrows, numbers 1–2) express Kdrl:nlsmCherry as they detach from the CVP (yellow arrows in j and teal pseudocolor in k).

Zebrafish FGPs are derived from kdrl-expressing endothelium.

(a-c) Lateral view confocal micrographs of a 5 dpf Tg(−9.8actb2:LOXP-DsRED-LOXP-EGFP); Tg(kdrl:Cre) double transgenic ‘switch’ embryo, with ‘unswitched’ DsRed-positive cells (magenta, panels a and b) and ‘switched’ EGFP-positive cells (green, panels a and c) where Kdrl:Cre has successfully excised the LoxP cassette. (d), Lateral view confocal micrograph of a 4 dpf Tg(-9.8actb2:LOXP-DsRED-LOXP-EGFP);Tg(kdrl:Cre) double transgenic embryo injected intravascularly with Qdot705 (red) to highlight all patent vessels. ‘Unswitched’ DsRed is in grey, and ‘switched’ EGFP is in green. (e) Higher magnification image of the yellow boxed area in panel d, showing an optic tectum vessel labeled with Qdot705 (red) surrounded by switched EGFP-positive FGPs (green). (f), Dorsal view confocal micrograph of the brain optic lobes of an adult Tg(−9.8actb2:LOXP-DsRED-LOXP-EGFP); Tg(kdrl:Cre) double transgenic ‘switch’ animal with mosaic expression of EGFP in a subset of FGPs. The white box notes the area shown in panel g. (g), Higher magnification image of the boxed region in panel f, with boxes noting the areas shown at still higher magnification in panels h and i. (h) Higher magnification confocal image showing autofluorescent green vesicles (yellow arrows) in unswitched FGPs. (i) Higher magnification confocal image showing EGFP-positive switched FGPs. Rostral is to the left in panels a-e, and up in panel f-i. Scale bars: 500 µm (a–c), 100 µm (g).

Mosaic expression of EGFP in vessel endothelial cells in the Kdrl:Cre ‘switch’ double transgenic line

(a,b) Lateral view confocal micrograph of the head of a 5 dpf Tg(−9.8actb2:LOXP-DsRED-LOXP-EGFP); Tg(kdrl:Cre) double transgenic ‘switch’ animal, showing DsRed-positive ‘unswitched’ cells (magenta, panel a) and mosaic EGFP expression in only a subset of ‘switched’ endothelial cells (green, panels a and b; arrows note switched EGFP positive endothelium). (c) Lateral view confocal micrograph of the head of a control 5 dpf Tg(kdrl:mCherry) animal, showing uniform mCherry transgene expression (grey) in all endothelial cells. (d-f) Higher magnification confocal micrographs of the same adult brain shown in Figure 9f, with arrows noting a partially switched vessel segment that is both DsRed positive (magenta, panels d and e) and EGFP positive (green, panels d and f).