Additional file 2: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Tutorial for the bioinformatics including sgRNA/primer deisgn and MiSeq analysis. (PDF 7778 kb

Additional file 4: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Amplicon screening data used for Fig. 4a-c. (TXT 142 kb

Additional file 7: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Code used to produce the plot in Figs. 4 and 5, Additional file 3: Figure S1. (TXT 14 kb

Additional file 5: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Results from KASP genotyping used for Fig. 4d. (TXT 910 bytes

Additional file 3: Figure S1 and Figure S2. of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Comparison of induced indels between germline and somatic tissues. (a) Plot shows the same data as Fig. 4a, split into separate plots for each sgRNA. (b) Plot of frequencies for individual variants in sperm versus fin clip (same data as in Fig. 4b with full axes). (PDF 374 kb

Transverse sections showing expression of <i>kiss-R2</i> messengers-expressing cells in the hypothalamus of sea bass.

<p>(A) <i>Kiss-R2</i> messengers expressing cells (arrowheads) in the nucleus anterioris tuberis (NAT) and the anterior nucleus lateralis tuberis (NLTa). Bar = 100 µm. (B–D) Strong expression of the <i>kiss-R2</i> mRNAs at different levels of the nucleus of the lateral recess (NRL) surrounding the lateral recess (rl). B: Bar = 75 µm; C: Bar = 75 µm; D Bar = 75 µm. (E–F) <i>Kiss-R2</i> messengers expressing cells (arrowheads) in the caudal nucleus lateralis tuberis (NLT) and in the nucleus of the posterior recess (NRP) surrounding the posterior recess (rp). E: Bar = 75 µm; F: Bar = 100µm.</p

Expression of <i>kiss-R2</i> mRNAs in chemically identified neurons in the brain of sea bass.

<p>Examples of <i>kiss-R2</i>-expressing cells exhibiting immunoreactivity to nNOS (neuronal nitric oxide synthase in A), NPY (neuropeptide Y in B), TH (tyrosine hydroxylase in C) and SRIF (somatostatin in D–G). A: Bar = 10 µm; B: Bar = 10 µm; C: Bar = 20 µm; D: Bar = 8 µm; D: Bar = 40 µm; E-G: Bar = 15 µm.</p

Detailed information on the plasmids and restriction enzymes.

<p>Detailed information on the plasmids and restriction enzymes.</p

Transverse sections showing examples of preproKiss2-immunoreactive structures in the brain of European sea bass.

<p>(A) Low power view at the level of the hypothalamus showing positive cells in the region of the nucleus of the lateral recess (arrows). Please note the high density of immunoreactive-fibers heading towards the dorsal diencephalon and the ventromedial hypothalamus (arrowheads). Bar = 100 µm; (B–C) High power view at the level of the hypothalamus showing positive cells in the region of the nucleus of the lateral recess showing in (B) many <i>kiss2</i> mRNAs detected by ISH and in (C), the same area stained with the Kiss2 antibody. D: Bar = 30 µm; D: Bar = 25 µm. (D–F) Examples of isolated bipolar Kiss2-immunoreactive neurons (D) adjacent to the horizontal commissure (HC), (E) above the caudal part of the lateral recess and (F) in the inferior lobe (IL). D: Bar = 10 µm; E: Bar = 40 µm; F: Bar = 40 µm. (G–I) Immunoreactive fibers in the dorsal (G; Vv) and ventral (H; Vv) portions of the subpallium and lateral to the preoptic area (I; POA). G: Bar = 30 µm; H: Bar = 40 µm; D: Bar = 75 µm<b>.</b> (J) Examples of cells receiving immunoreactive innervation in the anterior nucleus lateralis tuberis (aNLT). The cells are completely surrounded by immunoreactive profiles (arrows). (K) Positive fibers in the neurohypophysis (NH) at the level of the proximal pars distalis (PPD). D: Bar = 30 µm.</p

Distribution of preproKiss2-immunoreactive structures in the brain of the sea bass.

<p>(B,I) Shematic representation of preproKiss2-immunoreactive structures on representative transverse sections taken from the <i>Dicentrarchus labrax</i> brain atlas (Cerda-Reverter et al., 2001a, b; 2008). The level of these sections is shown in panel A taken from the atlas. Red circles represent the cell bodies and red dots the main fiber tracts. CCe corpus of the cerebellum; CE cerebellum; CP central posterior thalamic nucleus; Dlp lateral posterior part of the dorsal telencephalic area; Dm2 subdivision 2 of the medial dorsal telencephalic area; Dm3 subdivision 3 of the medial dorsal telencephalic area; Dp posterior portion of the dorsal telencephalon; E entopeduncular nucleus; HaCo habenular commissure; I intermediate thalamic nucleus; IL inferior lobe of the hypothalamus; MaOT marginal optic tract; NAPv anterior periventricular nucleus; NAT anterior tuberal nucleus; NC cortical nucleus; NDLIl lateral part of the diffuse nucleus; NDLIm medial part of the diffuse nucleus of the inferior lobe; NGT tertiary gustatory nucleus; NHd dorsal habenular nucleus; NHv ventral habenular nucleus; NLTI inferior part of the lateral tuberal nucleus; NLTm medial part of the lateral tuberal nucleus; NLTv ventral part of the lateral tuberal nucleus; nMLF nucleus of the medial longitudinal fasciculus; NPC central pretectal nucleus; NPGa anterior preglomerular nucleus; NPGc commissural preglomerural nucleus; NPGm medial preglomerural nucleus; NPOav anteroventral part of the parvocelullar preoptic nucleus; NPOpc parvocellular part of paraventricular organ; NPPv posterior periventricular nucleus; NPT posterior tuberal nucleus; NRLd dorsal part of the nucleus of the lateral recess; NRLl lateral part of the nucleus of the lateral recces; NRLv ventral part of the nucleus of the lateral recess; NRP nucleus of the posterior recces; NSC suprachiasmatic nucleus; LFB lateral forebrain bundle; OB olfactory bulbs; OC optic chiasm; OpN optic nerve; OT optic tectum; P pituitary; PCo posterior commissure; Pin pineal gland; PMgc gigantocellular part of the magnocellular preoptic nucleus; PMmc magnocellular part of the magnocellular preoptic nuycleus; PMpc parvocellular part of the magnocellular preoptic nucleus; PPd dorsal periventricular pretectal nucleus; PPv ventral periventricular pretectal nucleus; PSp parvocellular superficial pretectal nucleus; PVO paraventricular organ; SV saccus vasculosus; TEG tegmentum; TEL telencephalon; TLa nucleus of the torus lateralis; TLo torus longitudinalis; TPp periventricular nucleus of the posterior tuberculum; VCe valvula of the cerebellum; VL ventrolateral thalamic nucleus; VM ventromedial thalamic nucleus; VOT ventral optic tract; Vp postcommissural part of the ventral telencephalon. Scale bar = 1 mm.</p