PMC

Spatial expression of asip1 gene in zebrafish (A) and spotted gar (B). RT-PCR analysis of tissue specific expression pattern of asip1 in adults. Scale bar: (A)2 mm, (B) 1 cm.

Comparison of eGFP expression in rostro-caudal transverse sections (25 μm) from 150dpf Drer.asip1-iTol2-eGFP-BAC (A,C) and the Loc.asip1-iTol2-eGFP-BAC (B,D) transgenic zebrafish. Drer.asip1-iTol2-eGFP-BAC and Loc.asip1-iTol2-eGFP-BAC zebrafish show strong eGFP expression in the ventral skin region (dermis) and peritoneal membrane (C,D). Variegated eGFP fluorescence is seen in Loc.asip1-iTol2-eGFP-BAC dorsal dermis area (B). Cells that do not express eGFP are revealed with DAPI staining (blue color). Scale bars: 50 μm. d, dermis; ms, muscle; pc, peritoneal cavity; pm, peritoneal membrane.

Identification of zebrafish and spotted gar BACs likely to recapitulate asip1 expression. (A) T-coffee asip1 peptide sequence alignment of Drer.asip1 (ENSDART00000113083.2) and Loc.asip1. (ENSLOCT00000008713.1). Grey boxes show conserved amino acid residues. (B) Genomic organization and gene synteny comparison for genes annotated from zebrafish BAC clone (16N21) from DanioKey Library and spotted gar BAC clone (VMRC-56-161-M17) from BAC library VMRC-56 (Postlethwait Lab; Institute of Neuroscience, University of Oregon). Picture style was adapted from Genomicus [http://www.dyogen.ens.fr/genomicus-57.01/cgi-bin/search.pl]. Genes are depicted by colored polygons and transcriptional orientation is indicated at the angled end of each gene. Gene names are indicated above. Orthologs across the two species are depicted in the same colors. (C, D) Distribution of the spotted gar conserved coding and non-coding regions. Vista browser representation of the genomic region surrounding the spotted gar asip1 gene (LepOcu1, chr18:14032011-14456756), using Zv9 zebrafish genome (chr6:49873114-49991996) as reference. Conservation is represented as vertical peaks. Conserved non-coding elements (CNEs) and coding elements with possible enhancer activity are marked as red and blue bars, respectively.

BAC transgenes exhibit endogenous asip1-like expression in most tissues. Shown are representative eGFP fluorescence images in Drer.asip1-iTol2-eGFP-BAC (A,B,E,F,I,J,M,N)) and Loc.asip1-iTol2-eGFP-BAC (C,D,G,H,K,L,O,P) stable transgenic zebrafish lines. (A,C,I,K,M,O) are lateral views anterior to the left and (B,D,E,F,G,H,J,L,N,P) ventral views anterior to the left. eGFP fluorescence is first detected at 3dpf (A,B,C,D). (A, B) lateral and ventral views of eGFP expression in larval heads of Drer.asip1-iTol2-eGFP-BAC transgenic zebrafish showing specific expression restricted to the developing opercle. (C,D) lateral and ventral views of eGFP expression in larval heads of Loc.asip1-iTol2-eGFP-BAC transgenic zebrafish showing expression limited to the pectoral fins. (E–H) ventral views of eGFP expression in 30dph larvae, showing increased expression in different pharyngeal bone structures (opercle, dermal branchiostegal rays, quadrate dermal bone and pectoral fin folds) and in ventral skin area (white arrows) in Drer.asip1-iTol2-eGFP-BAC (E,F) and in the pectoral fin folds, interopercular bone, pectoral girdle and ventral skin area (white arrows) in Loc.asip1-iTol2-eGFP-BAC transgenic zebrafish line (G–H). (I–P) eGFP expression in 100dph fish, showing increased expression in the ventral skin region and the base of pectoral and pelvic fins of both BAC transgenic lines (M–P), eGFP was also detectable in different bone structures of pharyngeal region in both BAC Transgenic lines (I–L). Scale bars: (A–H) 200 μm; (I–P) 1mm. br, brachiostegals; e, eye; iop, interopercular bone; m, Meckel’s cartilage; op, operculum; pf, pectoral fin; pop, preopercle; pvf, pelvic fin; pg, pectoral girdle q, quadrate; 1D, 1V, 2V stripes.

Dorso-ventral gradient expression of asip1 in adult zebrafish (A) and spotted gar skin (C). Normalized gene expression levels of (B) zebrafish and (D) spotted gar asip1 in skin samples from belly to dorsum (locations of skin sample points are indicated in the picture). Shown are log 10-transformed ΔCt values of asip1 relative to eEf1α and β-actin, respectively. Data are the mean ±SEM from eight samples after triplicate PCR analysis. (E) Schematic representation of the rostro-caudal transverse sections. (F–I) Rostro-caudal transverse sections (12 μm thick) of paraffin embedded, in situ hybridization of asip1 expression in 210 dpf zebrafish (F,H) and 270dpf spotted gar (G,I). The sections in (H,I) shows the expression of asip1 transcripts in the dermis region of zebrafish (H) and spotted gar (I) ventral skin (black arrows). No aisp1 expression was found in the dermis region of dorsal skin of both species (C,D). Superscripts a, b, c and d indicate statistical differences (P<0.05) in gene expression levels among skin region (statistics data are similar if share at least one letter not sure what you mean). Scale bar: (A) 2 mm, (C) 1 cm, (F,G,H,I) 50 μm. d, dermis; m, muscle.

Confocal image projections of live Tg(Drer.asip1-iTol2-eGFP-BAC/sox10:mRFP) (A,C,E,G,H) and Tg(Loc.asip1-iTol2-eGFP-BAC/sox10:mRFP) (B,D,F) 5dpf larvae and 30dpf juvenile zebrafish. (A,B,C,D) Ventral view of a live 5 dpf larvae, anterior is to the left. sox10:mRFP specifically labels the pre- and post-migratory neural crest cells. (A,B) mRFP strongly labels the cartilages of maxillary, mandibular arches and pectoral fins. (A) Drer.asip1-iTol2-eGFP-BAC labels the developing opercle and dermal branchiostegal rays. (B) Loc.asip1-iTol2-eGFP-BAC labels the pectoral gridle. (A,B) The pectoral fin is labeled by both fluorescent proteins at 5 dpf. (C,D) Ventral view of a live 30dpf larvae heads, anterior is to the left. sox10:mRFP labels the cartilages of maxillary and mandibular arches. Drer.asip1-iTol2-eGFP-BAC labels different pharyngeal bone structures (interopercle, dermal branchiostegal rays, quadrate dermal bone) (C), and and Loc.asip1-iTol2-eGFP-BAC also labels some pharyngeal bone structures (interopercle and dermal branchiostegal rays) (D). (E,F) Ventral dermis region was strongly labeled by Drer.asip1-iTol2-eGFP-BAC and Loc.asip1-iTol2-eGFP-BAC. (G) Magnified view of ventral skin region from (E), showing dermis, peritoneal membrane and peritoneal cavity region. (H) Fluorescent dorso-ventral sagittal projection image of ventral skin area of 30 dpf juvenile showing clear sox10:mRFP expression in peritoneal membrane region where a reflecting iridophore layer is located. The peritoneal membrane region is also positive for Drer.asip1-iTol2-eGFP-BAC. However, the dermis region is exclusively labeled by Drer.asip1-iTol2-eGFP-BAC. Scale bar: (A,B) 100 μm, (C,D,E, F) 200 μm, (H,J) 50 μm. br, brachiostegals; ch, ceratohyal; cs, coraco scapular cartilage; d, dermis; e, eye; iop, interopercular bone; m, Meckel’s cartilage; ms, muscle; o, opercle; pc, peritoneal cavity; pf, pectoral fin; pg, pectoral gridle; pm, peritoneal membrane; pq, palatoquadrate; q, quadrate; sk, skin.