The reck  gene is expressed in neural crest derived and other tissue types during zebrafish

embryogenesis. (A) reck is expressed in the head and tail region of the embryos at Stage 12 H (12 hours post fertilization). (B) At stage

16H, reck is expressed in the optic vesicle, head mesenchymal cells, and somite mesoderm. (C) At stage 19 H, the reck gene is expressed

in a similar pattern with 16 H, but it is more evident in the ventral somites. (D) The reck expression is most evident in the head vasculature,

eye, and ventral vascular primordia at the 24 H stage. (E) At stage 48 H, the reck gene is mainly expressed in the head vascular vessels,

pharyngeal arches, and fin folds. (F) Higher magnification of the head region at stage 48 H (E). The reck expression is most evident in the

head vasculature and pectoral fin fold. ey, eye; hv, head vesicles; mfd, middle fin fold; op, optic vesicle; ot, otic vesicle; pa, pharyngeal

arches; pfd, pectoral fin fold; s, somite; tb, tail bud; vv, ventral vesicles.

(A–B) Loss of the reck gene function facilitates tumorigenesis in either rp or tp53 mutant background. Kaplan–Meier curves showing tumor-free survival of cohorts of single and double heterozygotes derived from rpL35^hi258/+ x sdp^w12/+ crosses (A) and tp53^M214K/+ x sdp^w12/+ crosses (B). Fish from all crosses were genotyped by PCR for each relevant mutation at 6–8 weeks of age, and housed segregated by genotype. In all panels, the numbers of fish (n) of each genotype are shown next to the genotype. The p values between the rp or tp53 single heterozygote and the compound mutants are shown in the figures. The green line (sdp^w12/+) and black line (non-carrier) are overlapped in panel (A). (C) More zebrafish developed pPNETs in the rpL35^hi258/+;sdp^w12/+ compared to rpL35^hi258/+. Two-tailed fisher's exact test was used for statistical analysis. p = 0.008. Asterisks indicate statistical significance (A–C). (D) A typical zebrafish with abdominal MNPST. The green arrow indicates the position of the tumor. (E) Hematoxylin and Eosin (H&E) staining showed typical histopathology of zebrafish MPNST, which is characterized by interlacing bundles and swirls of spindle shaped tumor cells. (F) A typical pPNET that was found in the tail region. The green arrow indicates the position of the tumor. (G) Representative examples of pPNET histology by H&E staining. The pseudorosettes (Flexner-Wintersteiner rosettes) are indicated with green arrows. (H) The wildtype allele is lost in some of the tp53^M214K/+;sdp^w12/+ tumors (tumor samples are labeled with asterisks). The reck gene was amplified by PCR using primers flanking the sdp^w12 mutation site. The PCR product was then digested with Ava II, which only cuts the 233bps wildtype PCR product into 158bps and 75bps fragments [34]. T, tumor; N, normal tissue from caudal fin. Note, the lowest band is from the PCR primer polymerization. Scaler bar = 250 mm (D, F) Scale bar = 50 mm (E, G).