Fig. 2

Generation of a fstl1a KO line. a) Diagram depicting the partial genomic structure of fstl1a showing the recognition site of gRNAs at exon 3 (grey bars) and of the generic primers used (arrows) for heteroduplex analysis and sequencing of the region. b) Low magnification images of embryos injected with fstl1a gRNA 1 and 2 showing examples of the distinct phenotypic classes. c) Plot of the percentage of embryos in each of the following phenotypic classes: wild type, dorsal curvature, dorso-ventral defects, severe defects, dead. Data from a single experiment; the total number of embryos is indicated in brackets. d) Gel image showing the amplification (by PCR) of the region flanking the PAM sequence using the generic primers mentioned in (a) from wild-type and Cas9 + gRNA1 injected embryos. e) Sequence alignments of the different mutations found in embryos derived from the cross of F0 individuals and wild types. The dashed rectangle indicates the allele selected for further work. bp: base pairs; ins: insertion; del: deletion; FS: frame shift; PSC: premature stop codon. f) Dot plot showing qPCR results for fstl1a and fstl1b in pooled wild-type and fstl1a KO 48hpf embryos from the third generation. Plot: technical replicates and mean ± CI (95%); NRQ: normalized relative quantity; (*) p = 0,007, t-test. g) Dot plot showing the quantification of the length of primary cilia in Kupffer?s vesicle of 12 somite-stage wild type and fstl1a mutant embryos. The pooled data from a single experiment are presented; the total number of cilia measured/number of embryos is indicated in brackets. Plot: individual length values and median ± CI (95%). (*) p = 3.8 × 10?14, Mann?Whitney test. h) Representative immunofluorescence images showing primary cilia (acetylated tubulin) in the nasal pit and otic vesicle of 48 hpf wild-type and fstl1a KO embryos. Scale bars: B: 1 mm; H: 20 µm.