Expression and developmental roles of esrp1 and esrp2 in zebrafish. a WMISH for esrp1 and esrp2 in Danio rerio WT embryos. At 14 h.p.f., esrp1 transcripts were observed in embryonic epidermis, while esrp2 expression was only detected in the polster (po). At 16 h.p.f., esrp1 was restricted to the posterior and tailbud (tb) epidermis, whereas esrp2 persisted in the hatching gland rudiment (hr) and mild expression started to be detected in the otic placode (ot). By 20 h.p.f., esrp1 was found in the tailbud epidermis and more subtly in the olfactory placode (ol), while esrp2 appeared in new territories, such as pronephros (pn) and ectodermal cells of tailbud fin fold (ff). At 24 h.p.f., expression of both paralogs presented a similar pattern including olfactory and otic placodes, cloaca (cl), and epidermis (ep), although esrp2 was also observed in the hatching gland (hg). By 36 h.p.f., both genes were detected in the inner ear epithelium (ie), notochord (nt), and phanynx (ph), and esrp2 was also observed in the heart (he). At 48 h.p.f., expression was found predominantly in inner ear and pharynx. b Schematic representation of the genomic and transcriptomic impact of the selected esrp1 and esrp2 mutations. Blue boxes/lines represent genomic deletions in the mutants, while the red line depicts an altered splice junction in the esrp1 mutant allele. TSS, transcription start site; PTC, premature termination codon; del, deletion. Standard and fluorescent (green dot) primers used during genotyping are represented by arrows. c Left: genotyping of embryos by fluorescent PCR readily distinguished between WT and MUT alleles. Right: Representative 5 d.p.f. larvae for wild type (WT), esrp1 mutant (esrp1 MUT/MUT), esrp2 mutant (esrp2 MUT/MUT), and double mutant (DMUT) genotypes. Deflated swim bladder in the DMUT embryo is indicated by a red asterisk. d–i Phenotypic differences between 6 d.p.f. WT (top) and DMUT (bottom) embryos in different embryonic structures. DMUT larvae showed impaired fin formation (arrows) and cleft palate (arrowhead) d, including malformation of the ethmoid bone, as shown by Alcian blue staining e. f–i Transversal histological sections stained with hematoxylin and eosin showing structural differences in pectoral fin f, esophagus g, inner ear h and olfactory epithelium i. Black arrowheads mark the dorso-lateral septum between semicircular canals in h. Proximal (pr) and distal (dl) parts of the fin are indicated in f. Scale bars: 1 mm a, 2 mm c–e, 100 µm f–h, 50 µm i
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