Figure 2.

tonsl^-/- Zebrafish Are Larval Lethal and Have Growth Deficits, Vertebral Abnormalities, and Reduced Neutrophil Numbers

(A) tonsl^−/− zebrafish are larval lethal and show progressively diminished size compared to that of wild-type (WT) siblings. Food intake is variable in mutants, and reduced food intake correlates with reduced fitness and increased mortality (the gut contents of larvae at 13 dpf are indicated with white arrows).

(B) tonsl^−/− fish (red) are not significantly smaller than WT siblings (blue) at 6 days post fertilization (dpf) or 8 dpf, but they are, on average, smaller at later time points through 13 dpf (N ≥ 10 larvae for each time point; p = 0.045 at 10 dpf; p < 0.0001 at 13 dpf). Normal zebrafish growth during this stage varies widely, and survivor bias is a factor in these data because tonsl^−/− mutants begin to die at 8 dpf.

(C) tonsl mutants exhibit precocious ossification of the axial skeleton. Bone formation is stained by Alizarin red, and cartilage is stained with Alcian blue. At 7 dpf, vertebral development is marked by bony centra forming around the notochord (asterisks). Significantly more centra have formed by this stage in homozygous tonsl mutants compared to WT siblings. WT—4.100 ± 0.5667, n = 10 larvae; tonsl^−/−:—8.867 ± 0.4350, n = 15 larvae.

(D) WT larvae have a high concentration of neutrophils in the gut (dashed outline), and neutrophils are dispersed throughout the circulatory system (D^o). mpo:gfp;tonsl^−/− mutants have variable neutrophil distribution that is correlated with their decline in health; the distribution ranges from normal (D′) to reduced neutrophil fluorescence in the gut (D″ and D″′), and from normal to diminished numbers of circulating neutrophils observable in blood vessels of the head and trunk (D″′).

(E) Compared to stage-matched WT zebrafish (blue) mpo:gfp;tonsl^−/−, mutants showing signs of decline (D″′, red) had a reduced number of circulating neutrophils. Gut neutrophils were excluded from this count (n = 10; p < 0.0001). Scale bars in A and D = 1 mm; in C = 500 nm. Student’s t tests with Welch’s correlation were performed for each dataset. Data in (B) are mean ± SD. Error bars indicate standard deviation.