The s364 phenotype is due to mutation in nsf. (A) Mutant and wild-type (WT) zebrafish (5 dpf) are shown on a white background. Mutant zebrafish display a dark appearance on the white background from dispersed melanin (red arrowhead). Wild-type zebrafish have an inflated swim bladder (black arrowhead) but mutant zebrafish do not. (B) Loss of pomc expression in the arcuate nucleus (ARC) of the hypothalamus (arrowheads) of mutant alleles but not wild-type at 5 dpf is shown. pomc expression is maintained in the anterior pituitary (ap) and posterior pituitary (pp) in both wild-type and mutant alleles. s364/st53 or s364/st25 transheterozygotes display the same pomc phenotype as s364, st53, and st25 homozygotes. Images are representative of at least three independent crosses of fifteen or more fish each. (C) Relative transcript levels are shown for nsf and nsfb in wild-type and st53 zebrafish heads at 5 dpf (n = 8 each). Fold induction is relative to one wild-type head for each primer set and taken to be 1.0 (horizontal dashed line). All other samples, including additional control and experimental samples, are normalized to this one wild-type sample with variation shown by error bars (standard error of the mean). (D) The linear growth of 5 dpf wild-type or st53 zebrafish is shown (wild-type = 3.6 mm, st53 = 3.8 mm; n = 10 each group). Length was measured from nose to tip of tail. Wild-type fish were temporarily paralyzed using ice. Results are expressed as mean ± standard deviation, and statistical analyses were done by unpaired t-test. ***P < 0.001. A schematic representation of NSF protein motifs. The st53 mutation introduces a nonsense mutation just prior to D2, whereas st25 introduces a missense mutation in D2. Two distinct amino-terminal domains (N1, N2) mediate substrate recognition.
|
