<i>Edin</i> expression is induced upon a wasp infection.

<p><b>(A)</b> Wasp infection causes a 6.7-fold increase in <i>edin</i> expression in 2^nd instar <i>Canton S</i> larvae. Data are pooled from two independent experiments, n = 2 for each experiment, where one sample represents 10 larvae. <b>(B)</b><i>Edin</i> expression is induced in the fat bodies of <i>Canton S</i> larvae 24 hours post infection. The data are pooled from four independent experiments, and each experiment consisted of two samples, where one sample represents 8–10 larval fat bodies.</p

A schematic presentation of the function of Edin.

<p>(1.) <i>Edin</i> is induced in the fat body shortly after wasp infection and secreted into the hemolymph. There, Edin directly or indirectly induces the release of plasmatocytes from the sessile hemocyte compartment. These cells go into circulation, find the wasp egg and participate in forming the capsule around the parasitoid egg. (2.) If the expression of <i>edin</i> is knocked down in the fat body in the context of a wasp infection, plasmatocytes are retained in the sessile compartment instead of being released into circulation, causing a defect in the encapsulation of the wasp egg.</p

<i>Edin</i> expression in fat body is dispensable for normal hemocyte attachment to and spreading on glass and wasp eggs, but is necessary to increase blood cell numbers in circulation early after wasp infection.

<p>(<b>A-B”</b>) Hemocytes from infected control larvae (<i>msnCherry</i>,<i>eaterGFP</i>, A-A”) and from infected larvae in which <i>edin</i> was knocked down in the fat body (<i>msnCherry</i>,<i>eaterGFP;Fb>edin</i>^{<i>109528</i>}, B-B”) spread normally on glass 14 hours after wasp infection despite knock down of <i>edin</i> in fat body. The spreading ability of hemocytes was assayed by staining α-Tubulin (blue) and F-actin (magenta). The size bar denotes 10 μm. (<b>C and D</b>). Wasp eggs from infected control larvae (<i>msnCherry</i>,<i>eaterGFP</i>, C) and from infected larvae in which <i>edin</i> was knocked down in the fat body (<i>msnCherry</i>,<i>eaterGFP;Fb>edin</i>^{<i>109528</i>}, D) were stained with the anti-plasmatocyte antibody <i>NimC1</i>. The wasp eggs were dissected 14 hours after parasitization and are still attached to the gut. Plasmatocytes spread normally on the eggs irrespective of <i>edin</i> RNAi in the fat body. Arrows denote examples of plasmatocytes spreading and adhering normally on the surface of the wasp egg. The scale bar depicts 50 μm. (<b>E</b>) <i>Edin</i> RNAi in the fat body (<i>msnCherry</i>,<i>eaterGFP;Fb>edin</i>^{<i>109528</i>}) reduced the number of circulating cells after wasp infection in comparison to control larvae (<i>msnCherry</i>,<i>eaterGFP</i>) 14 hours after infection. Circulating blood cell numbers were obtained with flow cytometry.</p

Knock down of <i>edin</i> in the fat body decreases the encapsulation and killing ability of <i>Drosophila</i> larvae.

<p><b>(A)</b> The encapsulation response of two different <i>edin</i> RNAi lines (<i>edin</i>^<i>14289</i> and <i>edin</i>^{<i>109528</i>}) was analyzed 27-29h after a wasp infection. The <i>C564</i>-<i>GAL4</i> (<i>C564>)</i>, <i>Fb-GAL4</i> (<i>Fb></i>) and <i>Hml</i>^<i>Δ</i>;<i>He-GAL4</i> (<i>HH></i>) drivers were used to drive the expression of the RNAi constructs. <i>w</i>^<i>1118</i> (<i>w</i>) was used as control. Data were pooled from one to eight individual experiments, as depicted on each column, each experiment with at least 50 analyzed individual infected larvae. <b>(B)</b> The ability of <i>Drosophila</i> larvae to kill wasp eggs was assessed with two different <i>edin</i> RNAi lines (<i>edin</i>^<i>14289</i> and <i>edin</i>^{<i>109528</i>}) 48-50h after infection. The <i>C564</i>-<i>GAL4</i> (<i>C564></i>) and <i>Fb-GAL4</i> (<i>Fb></i>) drivers were used to drive the expression of the RNAi constructs. <i>w</i>^<i>1118</i> (<i>w</i>) was used as control. Data are pooled from three to sixteen independent experiments, as indicated on each column, and at least 50 infected larvae were scored per experiment. Error bars in A and B show standard deviations. Knocking down the expression of <i>edin</i> in several tissues including the fat body or in the fat body alone caused a significant decrease in the encapsulation activity and killing response of <i>Drosophila</i> larvae compared to controls, whereas knocking down <i>edin</i> in hemocytes had no effect.</p

Quantification of hemocytes in <i>edin</i> RNAi larvae after a wasp infection.

<p><b>(A-B)</b> Hemocytes of infected larvae were bled 48–50 hours post-infection and visualized with the <i>eaterGFP</i> (green) and <i>msnCherry</i> (red) reporters. Uninfected controls contained only GFP-positive cells that corresponded to plasmatocytes (green). (<b>A’ and B’</b>) <i>msnCherry</i> expression was detected in the infected samples and this included lamellocytes (asterisks) and cells that express both <i>eaterGFP</i> and <i>msnCherry</i> indicating that they were undergoing lamellocyte transition. Lamellocytes were present also in the infected <i>edin</i> RNAi larvae suggesting that <i>edin</i> expression is not necessary for lamellocyte differentiation. Scale bars are 10 μm <b>(C-E)</b> Flow cytometry was carried out to quantify the amount of hemocytes in the unchallenged and the wasp infected <i>edin</i> RNAi larvae. (C = control, inf = infected)</p

Quantification of mycobacterial antigen expression with GFP ELISA.

<p>AB fish were immunized with 12 μg of experimental or control vaccine plasmids, followed by electroporation. Seven days post-injection, fish were dissected under a UV light and the dorsal muscles were collected and homogenized with ceramic beads, followed by protein extraction. 7.5–15 μg of each protein lysate in a 1% SDS buffer was used for a GFP ELISA analysis. A standard curve was used to quantify the absorbance values, which were then normalized with the average of the control values of each experiment before the values were pooled. Non-immunized AB fish were used as the negative control. Mean±SD is shown. N≥4 per group. * p<0.05, ** p<0.01, *** p<0.001 (Two-tailed Mann-Whitney test).</p

RpfE antigen improves survival of the fish infected with a high dose of <i>M</i>. <i>marinum</i>.

<p>AB fish were immunized intramuscularly with the experimental and control (GFP) antigens, followed by an intraperitoneal infection with ~10.000 cfu of <i>M</i>. <i>marinum</i>. Fish were then followed for 12 weeks for survival. The survival curve for each antigen immunization is shown separately with the GFP control group of the same infection experiment(s). ** p<0.01 (Log-rank (Mantel-Cox) test). N≥19 in each group.</p

Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (<i>Danio rerio</i>)

<div><p>CRISPR-Cas9 technology is routinely applied for targeted mutagenesis in model organisms and cell lines. Recent studies indicate that the prokaryotic CRISPR-Cas9 system is affected by eukaryotic chromatin structures. Here, we show that the likelihood of successful mutagenesis correlates with transcript levels during early development in zebrafish (<i>Danio rerio</i>) embryos. In an experimental setting, we found that guide RNAs differ in their onset of mutagenesis activity <i>in vivo</i>. Furthermore, some guide RNAs with high <i>in vitro</i> activity possessed poor mutagenesis activity <i>in vivo</i>, suggesting the presence of factors that limit the mutagenesis <i>in vivo</i>. Using open access datasets generated from early developmental stages of the zebrafish, and guide RNAs selected from the CRISPRz database, we provide further evidence for an association between gene expression during early development and the success of CRISPR-Cas9 mutagenesis in zebrafish embryos. In order to further inspect the effect of chromatin on CRISPR-Cas9 mutagenesis, we analysed the relationship of selected chromatin features on CRISPR-Cas9 mutagenesis efficiency using publicly available data from zebrafish embryos. We found a correlation between chromatin openness and the efficiency of CRISPR-Cas9 mutagenesis. These results indicate that CRISPR-Cas9 mutagenesis is influenced by chromatin accessibility in zebrafish embryos.</p></div

Correlation between mutagenesis efficiency and chromatin features at different developmental stages and timepoints.

<p>Correlation between mutagenesis efficiency and chromatin features at different developmental stages and timepoints.</p

Onset of mutagenesis differs between sgRNAs.

<p>Heteroduplex mobility assay to demonstrate the onset of mutagenesis using high efficiency guide RNAs targeting three different genes with different gene expression patterns in early development. Embryos were collected at timepoints 1, 2, 3, 4, 6hpf (15–20 embryos per group). The gene name above the gel image indicates CRISPR-Cas9 injected embryos and control indicates uninjected controls. The legend on the side indicates the positions of wt (wild type) and mutant bands in the gel. Red arrows indicate the point at which first mutations can be detected.</p