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First published online 23 March 2005
doi: 10.1242/dev.01791

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Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products

¹ Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA
² Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307 USA

View larger version (75K): [in a new window]   Fig. 1. Timecourse of zebrafish brain ventricle formation. (A-K) Ventricles were visualized by microinjecting a fluorescent dye, Texas Red-dextran, into the hindbrain ventricle of anesthetized embryos. (A) Ventricle injection schematic: lateral view of 24 hpf embryo with microinjection needle at injection site of hindbrain ventricle. (B-K) Developmental profile of brain ventricle morphology at 18, 20, 22, 24, and 30 hpf following dye injection; (B-F) dorsal views; (G-K) lateral views; anterior to left. Heartbeat onset at 24 hpf (E), after brain ventricles have formed. (L-O) Cell morphology of hindbrain ventricle was visualized by confocal microscopy after overnight immersion in fluorescent molecule bodipy ceramide. (L) Diagram of lateral view of 18 hpf embryo, with horizontal plane used for confocal time-lapse imaging indicated by green line. (M-O) Confocal time-lapse imaging of hindbrain ventricle of living, anesthetized embryo, beginning at 19 hpf and ending at 20 hpf. Asterisks label hinge-points from which opening begins, and arrows point to locations of apparent adhesion that release as ventricle opens anterior to posterior. Asterisks: midbrain and hindbrain hinge-points. Scale bar: 50 µm. A, anterior; F, forebrain ventricle; H, hindbrain ventricle; M, midbrain ventricle; P, posterior.

View larger version (101K): [in a new window]   Fig. 2. Initial brain ventricle formation is normal in the absence of circulation. Living, anesthetized embryos from a heterozygous cross of the silent heart (sih) mutant were injected with Texas Red-dextran. At 24 and 27 hpf, the pattern of ventricle formation is identical in wild type (A,C) and sih mutant (B,D). At 36 hpf, the volume of brain ventricles is smaller in sih (F) than in wild type (E). (A-D) Dorsal views, (E,F) side views, anterior to left. Scale bar: 50 µm.

View larger version (59K): [in a new window]   Fig. 3. Cell proliferation and cell death analysis in wild-type and mutant embryos. (A-C) Cell proliferation analysis, using PH3 antibody labeling. (A,B) Fixed and labeled wild-type brain at 17 and 21 hpf. (C) Quantification comparing MHB region and hindbrain, n=8, P=0.627 at 17 hpf; P=0.008 at 21 hpf. Cell proliferation at 21 hpf is significantly higher, showing an almost twofold increase in proliferation in the hindbrain than in the MHB. (D-G) Ventricle formation after inhibition of cell proliferation by aphidicolin treatment. (D,F) Live control and treated embryos after ventricle injection at 24 hpf; (E,G) same embryos as in D and F, fixed and labeled for cell proliferation. Reduced cell proliferation leads to a decrease in ventricle opening. (H-J) Cell death analysis, using TUNEL labeling with ApopTag kit. (H,I) Fixed and labeled wild-type brain at 17 and 21 hpf. (J) Quantification comparing midbrain, MHB and hindbrain regions, n=10, P=0.575 at 17 hpf; P=0.368 at 22 hpf. Error bars denote standard error. H, hindbrain ventricle; M, midbrain ventricle; MHB, midbrain-hindbrain boundary. Scale bar: 50 µm.

View larger version (44K): [in a new window]   Fig. 4. snk encodes the zebrafish Na+K+ ATPase Atp1a1a.1 protein. Predicted structure of the Na+K+ ATPase Atp1a1a.1 protein. Red dot in the M2-M3 loop represents site of amino acid change from glycine to aspartate at residue 271 in the snkto273a mutant. RT-PCR and sequencing was performed on the snk mutants from 3 snk carrier clutches (107 embryos), embryos from a snk sibling non-carrier clutch (76 embryos) and a wild-type clutch (79 embryos). In 100% of embryos, snk mutants, identified phenotypically, showed the G to A mutation, which would result in the glycine to aspartate amino acid change, whereas all wild-type embryos showed the normal G nucleotide.

View larger version (114K): [in a new window]   Fig. 5. Morphological analysis of snakehead and nagie oko brain ventricle mutants. (A-F) Light microscopy images of brain at 28 hpf. Dorsal views (A-C) or side views (D-F) of living, anesthetized embryos are shown, anterior to left. Ventricles are injected with Texas Red dextran in A. Relative to wild-type (A,D), brain ventricles in snakehead (B,E) and nagie oko (C,F) mutants appear to be absent: nok mutants sometimes form a small hindbrain ventricle that never enlarges. Note the characteristic refractivity of the neural tube in snk, in that neither the outline of the neural tube nor the brain folds are visible in the snk mutant (B,E). In addition, note that arrows in A and C point to the MHB constriction in wild type and nok, respectively. By light microscopy, the snk MHB constriction is not visible (even though it is in the proper location; see Q below). Bracket in D indicates hindbrain ventricle height. (G-O) Histology of snk and nok mutants. Embryos were fixed and transverse-sectioned at 22 hpf, at the level of forebrain, midbrain or hindbrain, and stained with hematoxylin and eosin. Relative to wild-type embryos (G,J,M), snk mutant embryos (H,K,N) show appropriate ventricle morphology; however, the cells appear to be adhered to one another and no lumen is present. By contrast, nok mutant embryos (I,L,O) fail to undergo any ventricle morphogenesis, and the epithelium appears disorganized. Asterisks label midbrain hinge-points in wild type (J) and snk (K), but hinge-points are absent in nok (L). (P-R) Confocal images through mid- and hindbrain ventricles of 24 hpf living embryos stained with bodipy ceramide. (P) Wild type, (Q) snk, (R) nok. Note that snk embryos (Q) assume correct ventricle morphology but fail to open the ventricles. By contrast, the brain tube in nok embryos (R) remains straight and no hinge-points form (although the MHB constriction remains). Scale bar: 50 µm. F, forebrain ventricle; H, hindbrain ventricle; M, midbrain ventricle. Asterisks: hinge-points.

View larger version (139K): [in a new window]   Fig. 6. Analysis of epithelial integrity in nok mutants. (A-L) Transverse sections through brains of wild-type and nok embryos at 17 hpf (A,B) or 22 hpf (C-L). (A,B) A clear midline is visible in wild type (A; arrow) but cells are disorganized in nok (B). (C,D) Labeled with PH3 antibody (mitosis marker) (green) and actin (red), dividing nuclei localize apically in both wild type (C) and mutant (D). (E,F) Labeled with ß-catenin antibody; (G,H) labeled with actin marker, phalloidin-Texas Red; (I,J) labeled with occludin antibody and phalloidin-Texas Red counterstain; (K,L) high magnification of 3D compilation of transverse sections through forebrain, labeled with phalloidin-Texas Red. In the nok mutant, all junction markers localize apically as in wild type but show disorganization. (M,N) Nok antibody labeling (green) at 22 hpf in horizontal section through midbrain and hindbrain of wild type (M) and nok mutant (N), with phalloidin-Texas Red as counterstain (red). Scale bar: 20 µm.

View larger version (71K): [in a new window]   Fig. 7. Epistasis analysis of brain ventricle mutants snk and nok. (A-H) Lateral and dorsal views of anesthetized, living embryos at 25 hpf. (A,E) Wild type (70/139 embryos, 50%); (B,F) nagie oko mutants (29/139 embryos, 21%); (C,G) snakehead mutants (30/139 embryos, 22%). (D,H) snakehead;nagie oko double mutants (10/139, 7%) show overall composite phenotype. Asterisks mark hinge-points, which are visible in wild type but not visible in the mutants (although hinge-points are present in the snk mutant; see K). Arrow marks midbrain-hindbrain constriction, which is visible in wild type and nok mutant but is not visible in snk nor snk;nok due to the altered refractivity of the snk tissue. (I-L) Histological sections through midbrain at 22 hpf of wild type (I), nok (J), snk (K) and snk;nok (L). The phenotype appears to be additive, suggesting nagie oko and snakehead function in separate pathways. (M-P) Drawings of brain outline and ventricle lumen in I-L show composite phenotype more clearly. Scale bar: 50 µm. H, hindbrain ventricle; M, midbrain ventricle.

View larger version (123K): [in a new window]   Fig. 8. -Na+K+ ATPase localization in nok mutants and Nok localization in snk mutants appears normal. (A,B) a6F antibody labeling (green) at 24 hpf in horizontal section through midbrain and hindbrain of wild type (A) and nok mutant (B) with phalloidin-Texas Red as counterstain (red). Note that while the nok mutant hindbrain ventricle is severely reduced, a6F antibody still labels apical membrane. (C,D) Nok antibody labeling (green) at 24 hpf in horizontal section through midbrain and hindbrain of wild type (C) and snk mutant (D), with phalloidin-Texas Red as counterstain (red). Nok localizes at the apical membrane in both wild type (C) and mutant (D), suggesting that Snk is not necessary for Nok targeting to apical surface. Note: the snk mutant does have normal hinge-points in the midbrain and hindbrain, although they are not present in the optical section shown in D. Scale bar: 50 µm.

View larger version (61K): [in a new window]   Fig. 9. Multiple steps are required for brain ventricle formation. Three steps have been identified during initial brain ventricle formation and occur independently of circulation. The Nagie oko protein helps maintain epithelial polarity and/or integrity, which is required for normal ventricle morphogenesis. Atp1a1a.1 is essential to inflate the ventricular space with fluid, and localized cell proliferation also appears to be necessary. Later brain ventricle expansion requires circulation. A, anterior; F, forebrain ventricle; H, hindbrain ventricle; M, midbrain ventricle; P, posterior.