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First published online March 31, 2005
Journal of Experimental Biology 208, 1537-1549 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01564

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Development of oxygen sensing in the gills of zebrafish

Michael G. Jonz^* and Colin A. Nurse

Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1

View larger version (51K): [in a new window]   Fig. 1. Neuroepithelial cells and associated innervation in the gills of adult zebrafish. (A) Confocal image of two adjacent gill filaments (f) with respiratory lamellae (lam). Neuroepithelial cells (NECs, green) were serotonin (5-HT)-immunoreactive (IR) and located in the filaments (i.e. O2-sensitive NECs) and lamellae (arrows). Both NEC populations were innervated by zn-12-IR nerve fibres (red; arrowheads) that emanated from nerve bundles. Scale bar, 50 µm. (B) Serotonin (5-HT)-immunoreactive (IR) NECs (green; arrows) of the gill arch (ga). NECs of the gill arch were organized along a zn-12-IR (red) nerve bundle (nb) and were innervated by zn-12-IR nerve fibres (arrowheads). Also visible are 5-HT-IR NECs of the respiratory lamellae (lam) near proximal regions of the gill filaments (f), and 5-HT-IR Merkel-like basal cells of the gill rakers (gr). Scale bar, 50 µm.

View larger version (82K): [in a new window]   Fig. 2. Gill arches and developing filaments in zebrafish larvae. Corresponding schematic drawings are shown below. (A) Phase-contrast image of the anterior end of a 3 d.p.f. larva showing the ventrally positioned gill arches from a lateral view. Scale bar, 100 µm. (B) Lateral view of developing gill filaments in a 5 d.p.f. larva. Same orientation as in A. Filaments are visible on gill arches 2, 3 and 4. In addition, red blood cells (arrowheads) were seen moving through filament primordia. Scale bar, 25 µm. (C) Ventral view of the left side of the gill basket of a 5 d.p.f. larva. The first three gill arches are visible and each gave rise to several filament primordia. Pigment of the overlying tissue partially obscures arch 3. Scale bar, 25 µm. Stippling in the diagram below indicates the space (i.e. buccal cavity) between gills. e, eye; f, filament; ga, gill arch; h, heart; ov, otic vesicle; p, pigment; ys, yolk sac.

View larger version (57K): [in a new window]   Fig. 3. Confocal image of an isolated zebrafish gill at 3 d.p.f. (A) Labelling with antibodies against serotonin (5-HT) demonstrated that O2-sensitive neuroepithelial cells (NECs) were absent in gill filament primordia (f), but two NECs (green) of an unidentified type were located in the gill arch (ga). Scale bar, 10 µm. (B) Image in A shown with zn-12-IR (red). 5-HT-immunoreactive (IR) NECs of the gill arch were associated with nerve fibres (arrows) of zn-12-IR nerve bundles (nb). Two zn-12-IR neurons (n) are also visible. Although NECs were not found in developing gill filaments at this stage, zn-12-IR nerve fibres (arrowheads) were present. (C) Higher magnification image of a single developing gill filament primordium (f) from another larva showing the absence of filament NECs and the presence of a zn-12-IR nerve fibre (arrowheads) as it entered the filament from the gill arch (ga). Scale bar, 5 µm. (D) Image in C shown with only zn-12-IR.

View larger version (34K): [in a new window]   Fig. 4. Innervation of neuroepithelial cells of the gill filaments in zebrafish larvae first appeared at 5 d.p.f. (A) Confocal image of an isolated gill from a 5 d.p.f. larva giving rise to four adjacent developing filaments (f). Serotonin (5-HT)-immunoreactive (IR) neuroepithelial cells (NECs, arrows) are shown in green and were present in gill filaments. 5-HT-IR NECs of the gill arch (ga) were also present. Scale bar, 10 µm. (B) Image in A shown with zn-12-IR. A zn-12-IR nerve fibre (arrowhead) is seen emanating from a nerve bundle (nb) of the gill arch in the direction of developing filaments but does not appear to make contact with filament NECs. In addition, 5-HT-IR NECs of the gill arch are shown associated with zn-12-IR nerve fibres. A zn-12-IR neuron (n) is also visible. (C) A developing gill filament (f) of another 5 d.p.f. larva contained a single 5-HT-IR NEC (green). Scale bar, 10 µm. (D) Image in C shown with zn-12-IR. Unlike the NECs depicted in A and B, this NEC was intimately associated with zn-12-IR nerve fibres (arrowheads) that arose from a nerve bundle (nb) of the gill arch (ga).

View larger version (35K): [in a new window]   Fig. 5. Innervation of neuroepithelial cells in developing gill filaments of 7 and 9 d.p.f. larvae. (A) Serotonin (5-HT)-immunoreactivity (IR) of an isolated gill from a larva at 7 d.p.f. At this stage, filaments (f) were longer and primordium of respiratory lamellae (lam) were observed. A single 5-HT-IR neuroepithelial cell (NEC, green) resided within the developing filament. Scale bar, 10 µm. (B) Image in A shown with zn-12-IR indicates that the NEC was associated with zn-12-IR nerve fibres (arrowheads) that arose from a nerve bundle (nb) of the gill arch (ga). A zn-12-IR neuron (n) is also visible. (C) 5-HT-IR of an isolated gill from a larva at 9 d.p.f. A 5-HT-IR NEC (green) of the filament (f) and a 5-HT-IR NEC of the gill arch (ga) were present. Scale bar, 10 µm. (D) Image in C shown with zn-12-IR reveals that both of these cells were associated with zn-12-IR (red) nerve fibres (arrowheads) arising from a nerve bundle (nb) of the gill arch (ga).

View larger version (28K): [in a new window]   Fig. 9. Chronology of the development of ventilatory events and structures in zebrafish. Solid arrows correspond to results obtained from the present study; dotted arrows indicate the work of previous authors. 1Kimmel et al., 1995; 2Rombough, 2002; 3Higashijima et al., 2000. 5-HT, serotonin; d.p.f., days postfertilization; IR, immunoreactive; NECs, neuroepithelial cells; HV, hyperventilatory

View larger version (11K): [in a new window]   Fig. 6. Effects of K+ channel blockers on ventilation frequency in adult zebrafish. Adults were immersed in control system water (Cont), 1 mmol l–1 quinidine (Quid), 25 mmol l–1 NaCl, or 20 mmol l–1 TEA + 5 mmol l–1 4-AP. Only quinidine induced a significant increase (asterisk) in ventilation frequency (mean ± S.E.M.; P<0.005; ANOVA–Bonferroni test). Sample sizes are indicated in the figure.

View larger version (12K): [in a new window]   Fig. 7. Effects of quinidine on ventilation frequency in adult zebrafish. (A) Zebrafish were exposed to 0.1 mmol l–1, 0.5 mmol l–1 and 1 mmol l–1 quinidine in a continuously perfused chamber. Bars represent the duration of quinidine application at each concentration. While 0.1 mmol l–1 quinidine had no effect, the addition of 0.5 mmol l–1 and 1 mmol l–1 significantly increased (asterisks) ventilation frequency (min–1; mean ± S.E.M.) compared to the control (N=6; P<0.05; ANOVA–Bonferroni test). This effect was fully reversible. (B) Dose–response curve of data in A. Data points from 2, 5, 8 and 11 min are plotted. Asterisks indicate a significant increase from control (0 mmol l–1). N=6; P<0.05; ANOVA–Bonferroni test.

View larger version (20K): [in a new window]   Fig. 8. Effects of hypoxia and quinidine on ventilation frequency in developing zebrafish. (A) Larvae 3–9 d.p.f. were subjected to control solution (Cont; PO2=150 mmHg; filled bars) or hypoxia (Hox; PO2=25 mmHg; open bars) in a continuously perfused chamber. Larvae responded to the hypoxic challenge with an increase in ventilation frequency (min–1; mean ± S.E.M.) as early as 3 d.p.f. This response increased to a maximum at 7 d.p.f. Asterisks denote a significant increase in ventilation frequency compared to control at each developmental stage (P<0.05; Student's t-test). In addition, the ventilatory response to hypoxia was significantly greater at 7 d.p.f. compared to the response at previous stages (P<0.001; ANOVA–Bonferroni test). Sample sizes are indicated in the figure. (B) Larvae 3–10 d.p.f. were exposed to control solution (Cont; filled bars) or 1 mmol l–1 quinidine (Quid; hatched bars) in the same perfusion system as in A. Ventilation frequency (mean ± S.E.M.) in 3 d.p.f. larvae was not significantly affected by the application of quinidine, but was quinidine-sensitive at 7 and 10 d.p.f. Asterisks indicate a significant difference from control at each developmental stage (P<0.05; ANOVA–Bonferroni test). Sample sizes are indicated in the figure.