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 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 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 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

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

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

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

Transverse sections showing expression of <i>kiss-R1</i> messengers-expressing cells in the brain and pituitary gland of sea bass.

<p>(A) Small <i>kissr1</i>-positive cells in the ventrodorsal (Vd) part of the telencephalon. Bar = 30 µm. (B–C) <i>kiss-R1</i> mRNA expression signal in the parvocellular preoptic nucleus (pars anteroventralis, NPOav) at low (B; Bar = 75 µm) and high magnification (C; Bar = 15 µm). (D) In the habenular region numerous <i>kiss-R1</i> expressing cells are identified especially in the ventral habenula (Hav). Bar = 50 µm. (E) Into the mediobasal hypothalamus, the medial part of the nucleus lateralis tubersi (NLTm) presents small clusters of <i>kiss-R1</i> mRNA expressing cells, just above the pituitary stalk. Bar = 25 µm. (F) <i>Kiss-R1</i> mRNA (arrows) are detected in the pituitary gland of sea bass, especially at the level of the proximal pars distalis (PPD). Scale bar = 100 µm.</p