Generation of nap1l1 transgenic line in zebrafish. (A) Sequence comparison of upstream intergenic sequences between the zebrafish nap1l1 gene with its human and mouse homologues (https://ecrbrowser.dcode.org). (B) The strategy for the target plasmid pDest-tol2-nap1l1-nEGFP construction with the primer positions of each reconstruction fragment. (C) Amplification of each reconstruction fragment. The DNA templates of lane 1?3 were plasmid pDest-tol2-pA2, zebrafish genomic DNA, and plasmid pME-nEGFP, respectively. Red arrowhead points to the right size of backbone fragment. (D) PCR-based Genotyping confirmed that the fluorescent or fluorescenceless F1 progeny harbor the Tg(nap1l1:nEGFP) transgene. Line1-2, the fluorescenceless F1 progeny of Tg(nap1l1:nEGFP)zs101 and Tg(nap1l1:nEGFP)zs102 respectively; Line3-6, the fluorescent F1 progeny of Tg(nap1l1:nEGFP)zs101, Tg(nap1l1:nEGFP)zs102, Tg(nap1l1:nEGFP)zs103, and Tg(nap1l1:nEGFP)zs104, respectively; Line7-8, each single wildtype embryo; Line9, zebrafish genome was not added as PCR template. (E-G, Ha, Ja-b, Ka-d) Overview of the transgene expression pattern of a zebrafish line Tg(nap1l1:nEGFP)zs102 during embryonic development. (Hb, Ia, Jc-e, Ke-f) In situ hybridization for egfp. (Hc, Ib, Jf-h, Kg-h) In situ hybridization for nap1l1. White arrowhead points to the embryonic shield. Blue or green arrowheads point to the lateral line neuromasts. Abbreviations: ALL, anterior lateral line; h, heart; nm, neuromast; nt, neural tube; pd, pronephric duct; PLL, posterior lateral line; ter, terminal neuromasts. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Early expression of nap1l1 transgenic line. Confocal imaging for the developmentally staged embryos or larvae from the out-cross of Tg(nap1l1:nEGFP)zs102 with the endothelial reporter line Tg(flk1:mCherry) and the neuronal reporter line Tg(huc:mCherry), respectively. (T) Confocal immunofluorescent imaging of the 52-hpf Tg(nap1l1:nEGFP)zs102 embryo with primary antibody MF20 (red). Inset demonstrates nap1l1-expressing cells within the developing heart (ventricle), which was analyzed by whole-mount in situ hybridization with antisense probe of nap1l1 mRNA. Abbreviations: ba, branchial arches; fb, forebrain; h, heart; hb, hindbrain; mb, midbrain; nm, neuromast; nt, neural tube; oc, optic cup; olv, olfactory vesicle; ov, otic vesicle; pav, paraxial vessel; v, ventricle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Adult expression of nap1l1 transgenic line. (A) Superficial overview or detailed observation of the adult male Tg(nap1l1:nEGFP)zs102. (A2,2?) Numerous rows of superficial neuromasts are situated on the skin surface, and each row of superficial neuromasts is called stitch. (B-E) Anatomical observation of the adult Tg(nap1l1:nEGFP;cmlc2:mCherry). (s6-20) Cryosection observation of anterior part of the adult Tg(nap1l1:nEGFP)zs102. Arrowhead in white points to a superficial neuromast, and arrowhead in yellow points to a canal neuromast. Abbreviations: CLL, caudal lateral line; io, infraorbital canal; po, preopercular canal; st, supratemporal canal; trunk, trunk canal. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)