Transgenic zebrafish (Gateways) expresses GFP in several CVOs and migratory microglia. (A–F) whole mounts (anterior to the left); (A,B) in vivo; (C,D) anti-GFP WISH (eyes and olfactory placode removed); (A–E) lateral view; (F) frontal view; (E,F) double immunohistochemistry (anti-GFP–green, anti-acetyl-tubulin–red; (E) eyes and olfactory placode removed), (G,H) anti-GFP immunohistochemistry on cross-sections. Scheme indicates the level of cross-sections shown in (G,H). ac, anterior commissure; ah, adenohypophysis; c, cerebellum; cpIII, choroid plexus of III ventricle; cpIV, choroid plexus of IV ventricle; d, diencephalon; e, epiphysis; h, hour postfertilization; hb, hindbrain; ht, hypothalamus; mhb, midbrain-hindbrain boundary; me, median eminence; mg, migratory microglia; oc, optic chiasm; ol, olfactory placode; on, optic nerve; ot, optic tectum; ovlt, organum vasculosum laminae terminalis; poc, postoptic commissure; t, telencephalon; tc, tela choroidea; #, subfornical organ. Scale bar−50 μm.

Four days old Gateways larvae express GFP in the OVLT. Confocal microscopy of compound transgenics Gateways (GFP, green)/Tg (kdrl:ras-Cherry) (red) in vivo reveals the OVLT in respect of developing vasculature. (A) Frontal view at the level immediately anterior to the optic chiasm; (B) dorsal view at the level of OVLT; (B') schema of the OVLT in respect of vasculature (based on the vasculature atlas, Isogai et al., 2001). cmv, communicating vessel; crdi, cranial division of the internal carotid artery; d, diencephalon; ht, hypothalamus; e, eye; mplv, median palatocerebral vein; oc, optic chiasm; ol, olfactory placode; on, optic nerve; ovlt, organum vasculosum laminae terminalis; pla, palatocerebral artery. Scale bar−50 μm.

Transgenic zebrafish lines express GFP in the AP as detected by GFP in vivo. Being a derivative of the roof plate, the AP is located at the dorsal hindbrain-spinal cord junction. In the Gateways transgenics the GFP expression domain in this area is rather broad unlike that in the ET33-B13 transgenics. All images (except I', which is a scheme based on I) are of whole mounts (anterior to the left). (A,D,E,H) lateral view, (B,C,F,G,I,I') dorsal view. (A–D,H,I) in vivo whole mounts. ap, area postrema; cpIII, choroid plexus of III ventricle; cpIV, choroid plexus of IV ventricle; dlav, Dorsal longitudinal anastomotic vessel; h, hour postfertilization; hb, hindbrain; ol, olfactory placode; ot, optic tectum; r5, rhombomere 5; se, intersegmental vessel; IVv, IV ventricle. Scale bar−50 μm.

Gateways express GFP in the developing hypothalamus. Cross-sections of Gateways larvae stained by immunohistochemistry. The ME is in the ventral midline position in contrast to the PVO, which is more dorsal and in lateral walls of hypothalamus. Scheme indicates the level of cross-sections shown in (A,B). (A,B) Cross-sections, (A) anti-GFP; (B) anti-GFP (green)/anti-acetyl-tubulin (red). Scale bar−50 μm.

Gateways and ET22-1 transgenics express GFP in the developing epiphysis (pineal). (A,B) Gateways; (C,D) ET22-1. (A–C) whole mounts, (D) cross-section. (A) Dorsal view; (B) frontal view (superficial scan); (C) lateral view; (A–C) in vivo, (C) anti-GFP immunohistochemistry. e, epiphysis; h, hour postfertilization; lcpIV, lateral cluster choroid plexus of IV ventricle; m, mouth; mcpIV, medial cluster, choroid plexus of IV ventricle; op, olfactory pit; ov, otic vesicle; pp, parapineal. Scale bar−50 μm (A,B), 25 μm (C), 10 μm (D).

Transgenic zebrafish lines (ET33-10, ET27) express GFP in the SCO, which represents the anterior bulging of the midbrain roof plate. The SCO expression detected by WISH (magenta, A) or by confocal microscopy in vivo (green-B,B'). Sco-spondin forms the Reissner fiber detected by anti-Reissner fiber antibody (red, B'). All images are of whole mounts in lateral view (anterior to the left). (A) WISH; (B) confocal microscopy in vivo; (B') two-color immunohistochemistry; (A) ET33-10; (B,B') ET27. cpIII, choroid plexus (IIIrd ventricle); d, diencephalon; e, epiphysis (pineal); h, hour postfertilization; mhb, midbrain-hindbrain boundary; mrp, midbrain roof plate; ot, optic tectum; rf, Reissner fiber; sco, subcommissural organ; t, telencephalon. Scale bar−50 μm.

Four days old ET33-mi2A zebrafish larva express GFP in the SVO. Confocal light sheet microscopy of compound transgenics ET33-mi2A (GFP, green)/Tg (kdrl:ras-Cherry) (red) in vivo reveals the SCO in respect of developing vasculature. (A) lateral midline view; (B) dorsal view; (B') schema of the relative position of the SCO in respect of vasculature (based on the vasculature atlas, Isogai et al., 2001). acev, anterior (rostral) cerebral vein; d, diencephalon; e, epiphysis; mmcta, middle mesencephalic central artery; mrp, midbrain roof plate; ot, optic tectum; pra, prosencephalic artery; sco, subcommissural organ. Scale bar−50 μm.

Schematic of CVOs detected using ET transgenics in 4 days old zebrafish brain (96 hpf). ac, anterior commissure; ah, adenohypophysis; ap, area postrema; asterisk, ovlt (organum vasculosum of the lamina terminalis); c, cerebellum; cf, cephalic flexure; cpIII, choroid plexus of III ventricle; cpIV, choroid plexus of IV ventricle; d, diencephalon; h, hindbrain; ht, hypothalamus; mhb, midbrain-hindbrain boundary; oc, optic chiasm; ot, optic tectum; pc, posterior commissure; pin, pineal gland; poc, post optic commissure; pp, parapineal gland; r5, rhombomere 5; sco, subcommissural organ; t, telencephalon.

Mutant analysis of development of the OVLT and SFO at 48 hpf. (A,B) Frontal view and confocal cross-section of control embryo. Strong GFP expression maps the OVLT-SFO complex, which clearly separates into two parts–dorsal and ventral (white arrows). (C,D) Frontal view and confocal cross-section of ace^−/− mutant embryo. The GFP expression domain corresponding to the OVLT/SFO complex not detected (highlighted with circle). (E,F) Ventral view and confocal section of the mib^−/− mutant. The dorsal part of this complex is missing. (G,H) Ventral view and confocal section of MBL mutant, respectively. Absent of expression at region of OVLT (highlighted with circle). (I,J) Ventral view and confocal section of SMU mutant, respectively. Absence of OVLT domain at 48 hpf (highlighted with circle). For confocal sections, the left part represents the optical cross section and right part-saggital section. Red line through cross section indicates where saggital section is. Blue line through saggital section indicates, where cross-section is. All images are taken with 25x magnification with no zoom except mib and smu mutants, which is taken with 10x magnification with 2x zoom. Abbreviations: op, olfactory pits; ovlt, organum vasculosum of lamina terminalis. Scale bar−50 μm.

Transgenic zebrafish (Gateways) expresses GFP in the AP, OVLT, SFO, ME, PVO, and migratory microglia. (A) anti-GFP WISH and (B) immunohistochemistry. (A,B) wholemount lateral view (eyes removed); anterior to the left. ah, adenohypophysis; ap, area postrema; c, cerebellum; cpIII, choroid plexus of III ventricle; cpIV, choroid plexus of IV ventricle; d, diencephalon; e, epiphysis; h, hour postfertilization; hb, hindbrain; ht, hypothalamus; mhb, midbrain-hindbrain boundary; me, median eminence; ot, optic tectum; ovlt, organum vasculosum laminae terminalis; pt, posterior tuberculum; pvo, paraventricular organ; r5, rhombomere 5; t, telencephalon, #, SFO. Scale bar 50–μm. *Defines clusters of cells expressing GFP mRNA.

The expression pattern of memKR in KR19 mimics that of GFP in Gateways. ap, area postrema; cp, choroid plexus; pin, pineal; rp, roof plate. Scale bar−50 μm.