The transcript distribution of the chemokine <i>sdf1a</i> is anteriorly extended in the <i>nar</i> mutant somite at st. 27.

<p>(A-H) Whole mount <i>in situ</i> hybridization using an antisense probe specific to the <i>sdf1a</i> ORF (A-D, <i>sdf1a-orf</i>) or distal 3'UTR (E-H, <i>sdf1a-distal</i>) at st. 27. Wild-type (A, B, E, F) and <i>nar</i> homozygote (C, D, G, H) embryos were stained using each probe. The <i>sdf1a</i> signals are observed in the mesodermal tissue corresponding to the gathering PGC region (B, D, F, bracket). The <i>sdf1a</i> signals in the ventrolateral somites (ls) are extended more to the anterior side than that in the wild type in the <i>nar</i> mutant embryos (C, D, dashed lines) using the ORF specific probe. The <i>sdf1a</i> signal is hardly detected in the <i>nar</i> mutant embryo using an <i>sdf1a</i> distal-3'UTR specific probe (G, H). Dorsal (A, C, E, G) and the lateral (B, D, F, H) views are shown in the orientation of anterior toward the top. AB, CD, EF, GH are the same embryos. Scale bars indicate 100 μm.</p

Characterization of the <i>naruto</i>^{<i>j113-2B</i>} (<i>nar</i>^{<i>j113-2B</i>}) mutation in medaka.

<p>(A-F) Altered PGC distributions in the <i>nar</i> mutants. PGCs were labeled by <i>in situ</i> hybridization with a medaka <i>vasa</i> (<i>olvas</i>) probe. At st. 22, PGCs are aligned bilaterally in the posterior trunk region in the <i>nar</i> mutant embryo (B, brackets) similar to the wild type (A, brackets). In the wild-type embryo at st.27, PGCs are clustered at the ventrolateral region of the prospective gonad (C, brackets), in contrast to the PGCs which are scattered anterior and to the lateral sides of the normal clustering position on the yolk sac (D, underlined by dashed lines) of the <i>nar</i> mutant. PGCs are gathered in the medial areas in the wild-type embryos at st. 31 (E, brackets). In the <i>nar</i> mutant embryos at the same stage (F), a part of the PGCs are gathered in the normal position (brackets), whereas some PGCs are scattered in the ectopic area (arrowheads). (G-H) Whole mount <i>in situ</i> hybridization of <i>cxcr4b</i> in wild-type (G) and <i>nar</i> (H) embryos at st. 27. The <i>cxcr4b</i> transcript distribution is observed in posterior lateral line primordia (pllp). The posterior lateral line primordia are distributed remarkably anterior in the <i>nar</i> mutant embryo (H) compared to the wild-type embryo (G). Weak <i>cxcr4b</i> transcript distribution is also seen in PGCs in the wild type (G, bracket) and is more laterally positioned in the <i>nar</i> mutant embryo (H, arrowheads). (I-P) Pictures of the live embryos showing the <i>nar</i> morphogenetic defects and their normal siblings. The <i>nar</i> mutant embryos exhibit enlarged brain ventricles (bv) at st.27 (I-L), a curved trunk, an enlarged pericardial space (ps) and a thin heart (h) at st. 35 (M-P). After st. 27 (N), blood circulation is stopped and clogged blood (cb) is observed. Dorsal views are shown in the orientation of anterior toward the top in the A-J, M-N. Lateral views are shown in the orientation of anterior toward the top in the K-L, O-P. Flat-mount embryos at st. 22 are shown in A-B. Scale bars indicate 100 μm.</p

Number of embryos injected with the <i>cpsf6</i> mRNA.

<p>Number of embryos injected with the <i>cpsf6</i> mRNA.</p

Mutation in <i>cpsf6/CFIm68</i> (<i>Cleavage and Polyadenylation Specificity Factor Subunit 6</i>) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant <i>naruto</i>

<div><p>Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified <i>cxcr4b</i> and <i>cxcr7</i>, which are both receptors of the chemokine <i>sdf1/cxcl12</i>, as key regulators of PGC migration. Among PGC migration mutants, <i>naruto</i> (<i>nar</i>) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the <i>nar</i> gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of <i>sdf1a/b</i> and <i>cxcr7</i> transcripts in the mutant embryos indicated shorter 3’UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3’UTR cleavage. In addition, expression of the coding region sequences of <i>sdf1a/b</i> in <i>nar</i> mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in <i>nar</i> mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3’UTR sequence. The abnormal expression pattern of <i>sdf1a</i> coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of <i>nar</i> mutation, further analysis using the <i>nar</i> mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes <i>in vivo</i>.</p></div

Identification of the <i>nar</i>^{<i>j113-2B</i>} mutation in a <i>cpsf6</i> gene.

<p>(A) Genetic linkages between polymorphic markers on chromosome 23 and the <i>nar</i>^{<i>j113-2B</i>} mutation. Recombination frequencies at the markers are indicated on the top of the vertical solid lines indicating each marker position on the chromosome 23 (horizontal bar). The <i>nar</i>^{<i>j113-2B</i>} was mapped between the <i>erc1</i> and the <i>smo</i> loci on chromosome 23 using a 1716 recombination density F2 panel. Further fine mapping using 5 or 9 recombinants at the <i>erc1</i> or the <i>smo</i> loci respectively resulted in narrowing down (horizontal black arrows) the <i>nar</i> candidate locus to within the 33kb region (double-headed horizontal red-arrow) containing the <i>cpsf6</i>. The chromosome information with annotated genes is referred from the Ensembl <i>Oryzias latipes</i> release version 63.1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172467#pone.0172467.ref080" target="_blank">80</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172467#pone.0172467.ref081" target="_blank">81</a>]. The individual contigs that make up the genomic assembly are colored in light or dark blue, and the gaps on the genome sequence are colored in white. The arrows of the annotated genes on the genome indicate the direction of the genes. (B) A local genome-sequence data set comparing the wild type (+/+) and <i>nar</i>^{<i>j113-2B</i>} homozygotes (-/-) demonstrating the T to A mutation at a splice donor site of the exon 2 in the <i>cpsf6</i> in the <i>nar</i>^{<i>j113-2B</i>} mutants. This mutation creates a <i>MnlI</i> site and an additional seven amino acids with a stop codon (red letters in the amino acid codes) from W⁹⁰ in the expected CPSF6 amino acid sequence. (C) Predicted wild-type CPSF6 (upper) is 551 amino-acid length, contains highly conserved domains, RNA recognition motif (RPM), proline rich domain (Pro-rich) and charged domain (CD). Whereas, the predicted CPSF6 in the <i>nar</i> mutant embryos is 97 amino-acid length lacking almost all conserved domains (lower). (D) Genomic DNA structure of medaka <i>cpsf6</i> exon2-6. The numbers under the exons (rectangles) and introns (solid lines) indicate the DNA length (bp) in each region. (E) Electropherogram of RT-PCR amplified from total RNA of wild-type (+/+) or <i>nar</i> mutant (-/-) embryos at st.27 using primers indicated in D (arrows). This image was made by the automated microchip electrophoresis-system. The PCR product in the mutant is longer corresponding to the intron size between the exons 2 and 3 (115bp) than in the wild type. Mw: molecular weight marker, phiX174 DNA-<i>HaeIII</i> digest, with the sizes (bp) indicated on the left. (F-I) Whole-mount <i>in situ</i> hybridization using a <i>cpsf6</i> antisense probe corresponding to the open reading frame. At st. 22, <i>nar</i> mutants (F) have strong ubiquitous <i>cpsf6</i> transcript distribution in their whole body similar to wild type (G). The ubiquitous <i>cpsf6</i> transcript distribution continued in wild type at st. 27 (H), whereas it became weak in the mutant embryo (I). F, G: flat mounted embryos. Dorsal views are shown in the orientation of anterior toward the top. Scale bar, 100 μm.</p

Poly A addition occurred unusually upstream and that caused short 3'UTRs in the <i>nar</i> mutant embryos.

<p>3' UTRs of the <i>sdf1a</i>, <i>sdf1b</i> and <i>cxcr7</i> were amplified by 3' RACE PCR from the total RNA of wild-type (wt) and <i>nar</i>^{<i>j113-2B</i>} homozygote (<i>nar</i>) embryos at st. 27, and separated by agarose gel electrophoresis. Short 3'UTRs (red) are detected in the <i>nar</i> mutant embryos in addition to 3'UTRs the same length as the wild type. The lengths of the 3'UTRs were determined by sequencing of cDNAs purified from each excised band. Actual sizes of each band are as follows, <i>sdf1a</i>: 1500, 600bp; <i>sdf1b</i>: 2600, 1600, 550bp; <i>cxcr7</i>: 2000, 800, 500bp. Upper and lower ends of the gel-images correspond to 4kb and 400bp respectively. Concentration of the genomic-DNA templates is not adjusted equally between the wild-type and mutant embryos.</p

The 3' end 100b sequences of 3'UTRs amplified by 3'RACE.

<p>The 3' end 100b sequences of 3'UTRs amplified by 3'RACE.</p

Additional file 1: Table S1. of Highly efficient generation of knock-in transgenic medaka by CRISPR/Cas9-mediated genome engineering

Genomic location of potential off-target sites for sgT (sgRNA for Tbait) (DOCX 14 kb

Additional file 1: Table S1. of Highly efficient generation of knock-in transgenic medaka by CRISPR/Cas9-mediated genome engineering

Genomic location of potential off-target sites for sgT (sgRNA for Tbait) (DOCX 14 kb

Additional file 3: Figure S1. of Highly efficient generation of knock-in transgenic medaka by CRISPR/Cas9-mediated genome engineering

GFP expression in Tg[pax7a-hs:GFP] and Tg[sox5-hs:GFP]. (A, B) Tg[pax7a-hs:GFP]. (C, D) Tg[sox5-hs:GFP]. (A, C) Dorsal views of the head region magnified. (B, D) Lateral views of the anterior trunk region magnified. The signal in the tectum is especially strong in Tg[pax7a-hs:GFP] (A). Presumable pigment cell progenitors of xanthophore and leucophore (B) and xanthophore (D) on the body surface are positive for GFP in Tg[pax7a-hs:GFP] and Tg[sox5-hs:GFP], respectively. (JPEG 1442 kb