Metronidazole-dependent loss of eGFP fluorescence indicates an effect on <i>r-opsin1+</i> PRCs in transgenic animals.

<p>All images show live immature adult <i>r-opsin1::egfp-f2a-ntr</i> transgenic animals (10-20 segments). (A-D) Concentration- and incubation time-dependent loss of eGFP fluorescent cells from adult eyes and frontolateral eyelets. Animals were treated with DMSO (A-A”) or varying concentrations of metronidazole (B-B”: 7mM, C-C”: 10mM; D-D”: 12mM). Duration and type of treatment are indicated on top of each panel. Arrowheads point at the axonal projection from the adult eye photoreceptors. Asterisks: head pigment cells that show autofluorescence in the channel used for eGFP documentation. These pigment cells expand or contract depending on light intensity and time of day. Arrows indicate adult eye pigments (panels A-D) position of eGFP-expressing PRCs (panel A’), position of lateral frontal eyelets (A”-D”). (E,F) Loss of eGFP fluorescent non-cephalic PRC upon metronidazole treatment. (E) <i>Parapodium</i> with eGFP-expressing non-cephalic PRC (arrow) and its axonal projection (arrowhead) before metronidazole treatment; (F) same specimen after 48 hrs of exposure to 12mM mtz, with arrow and arrowhead indicating the same positions. Scale bars: 50µm.</p

Loss of <i>r-opsin1+</i> cells by metronidazole treatment.

<p>Whole mount <i>in </i><i>situ</i> hybridization with a riboprobe specific for <i>Platynereis </i><i>r-opsin1</i> on immature adult <i>Platynereis</i> worms (10-20 segments). (A,C,E) DMSO controls, (B,D,F) metronidazole treated worms (12mM, 48hrs). (A,B) Dorsal views on heads (anterior down), focused on the position of the lateral frontal eyelets (arrows). (C-F) Ventral views (anterior down) on immature adult worm tails (C,E) treated with DMSO, (D,F) treated with mtz. Arrows point at the position of the peripheral <i>r-opsin1+</i> photoreceptor cells. Black ‘needle-like’ structures in C-F are aciculae (bristles). Scale bar: 30µm.</p

Metronidazole treatment specifically ablates <i>ntr</i> expressing cells, without affecting other brain cells.

<p>(A-F) Metronidazole treatment has no effect on non-PRC marker genes. Comparative analysis of expression patterns of the neuronal marker genes <i>prohormone </i><i>convertase2/phc2</i> (arrowheads in A-C) and <i>tyrosine </i><i>hydroxylase/th</i> (arrowheads in D-F) in untreated (A,A’,D,D’), DMSO treated (B,B’,E,E’) and metronidazole treated (C,C’,F,F’) animals. Each set of panels compares non-transgenic control animals (left) and <i>r-opsin1::eGFP-f2A-ntr</i> transgenic animals. Neuronal marker genes are detected in blue, <i>r-opsin1</i> were detected with FastRed substrate (red). In panels (A’-F’), FastRed is visualized using fluorescence microscopy. Scale bar: 50µm. (G,H) Quantification of cell numbers in untreated, DMSO treated and mtz treated animals. Individual eGFP fluorescent PRCs (green bars) were counted in live transgenic worms (same animals were counted before and after treatment). <i>phc2</i> (G) and <i>th</i> (H) expressing cells (black bars) were determined by counting all cells that showed complete cellular outlines in WMISH analyses of animals fixed after the respective experiment. Data represent means ± S.E.M. (n=10 worms for each experiment). ****<i>p</i><0.0001; ns. - no statistically significant differences. The two-tailed paired student <i>t</i>-test was used for statistical analyses.</p

Conditional and Specific Cell Ablation in the Marine Annelid <i>Platynereis dumerilii</i>

<div><p>The marine annelid Platynereis dumerilii has become a model system for evo-devo, neurobiology and marine biology. The functional assessment of its cell types, however, has so far been very limited. Here we report on the establishment of a generally applicable, cell type specific ablation technique to overcome this restriction. Using a transgenic strain expressing the bacterial enzyme nitroreductase (ntr) under the control of the worm’s r-opsin1 locus, we show that the demarcated photoreceptor cells can be specifically ablated by the addition of the prodrug metronidazole (mtz). TUNEL staining indicates that ntr expressing cells undergo apoptotic cell death. As we used a transgenic strain co-expressing ntr with enhanced green fluorescent protein (egfp) coding sequence, we were able to validate the ablation of photoreceptors not only in fixed tissue, using r-opsin1 riboprobes, but also by monitoring eGFP+ cells in live animals. The specificity of the ablation was demonstrated by the normal presence of the eye pigment cells, as well as of neuronal markers expressed in other cells of the brain, such as phc2, tyrosine hydroxylase and brn1/2/4. Additional analyses of the position of DAPI stained nuclei, the brain’s overall neuronal scaffold, as well as the positions and projections of serotonergic neurons further confirmed that mtz treatment did not induce general abnormalities in the worm’s brain. As the prodrug is administered by adding it to the water, targeted ablation of specific cell types can be achieved throughout the life of the animal. We show that ablation conditions need to be adjusted to the size of the worms, likely due to differences in the penetration of the prodrug, and establish ablation conditions for worms containing 10 to 55 segments. Our results establish mtz/ntr mediated conditional cell ablation as a powerful functional tool in Platynereis.</p> </div

Tol2-mediated transgenesis with pTol2{rps9::egfp} yields robust, ubiquitous expression of EGFP in G₀ animals.

<p>(<b>A, B</b>) pTol2{rps9::egfp}^frkt868-injected animals co-injected with <i>tol2</i> mRNA (A) show significantly stronger EGFP fluorescence (green arrows) than those without transposase (B) (ae: autofluorescence around the adult eyes). (<b>C, D</b>) Stable ubiquitous EGFP expression (green arrows) two months after co-injection of donor DNA and <i>tol2</i> mRNA (C) compared to a non-injected animal (D). Labels indicate autofluorescence of the jaws (j) as well as the iridophore pigments (p) in both the head and trunk.</p

Construction and expression of the <i>r-opsin1::egfp-f2a-ntr transgene</i>.

<p>(A) Schematized generation of the <i>r-opsin1::egfp-f2a-ntr</i> construct. The <i>egfp-f2a-ntr</i> cassette was recombined into the <i>Platynereis </i><i>r-opsin1</i> locus by homologous recombination. 8kbps of the surrounding genomic locus plus cassette were PCR amplified and subcloned into the mariner transposon vector used for transgenesis. (B-D) Co-expression of <i>egfp</i> and <i>ntr</i> in the adult eye photoreceptors of <i>r-opsin1::egfp-f2a-ntr</i> stable transgenic worms. (B) eGFP fluorescence demarcating the adult eye photoreceptors and their projections in stable <i>r-opsin1::egfp-f2a-ntr</i> transgenic worms. (C) Expression of <i>nitroreductase</i> (red) in the same cell type as visualized by whole mount in situ hybridization (WMISH) using <i>nitroreductase</i> antisense riboprobe. (D) Co-staining with <i>nitroreductase</i> (detected in red) and <i>egfp</i> (detected in blue) riboprobes results in purple color, indicative of faithful co-expression of both genes in the adult eye photoreceptors. ae- adult eyes; arrowheads point at expressing cells; arrow points at axonal projection of PRCs; asterisks- head pigment cells which show autofluorescence in the channel used for eGFP documentation. Scale bar: 20µm.</p

Tools for Gene-Regulatory Analyses in the Marine Annelid <i>Platynereis dumerilii</i>

<div><p>The advent of high-throughput sequencing technology facilitates the exploration of a variety of reference species outside the few established molecular genetic model systems. Bioinformatic and gene expression analyses provide new ways for comparative analyses between species, for instance, in the field of evolution and development. Despite these advances, a critical bottleneck for the exploration of new model species remains the establishment of functional tools, such as the ability to experimentally express genes in specific cells of an organism. We recently established a first transgenic strain of the annelid <i>Platynereis</i>, using a Tc1/mariner-type Mos1 transposon vector. Here, we compare Mos1 with Tol2, a member of the hAT family of transposons. In <i>Platynereis</i>, Tol2-based constructs showed a higher frequency of nuclear genome insertion and sustained gene expression in the G0 generation. However, in contrast to Mos1-mediated transgenes, Tol2-mediated insertions failed to retain fluorescence in the G1 generation, suggesting a germ line-based silencing mechanism. Furthermore, we present three novel expression constructs that were generated by a simple fusion-PCR approach and allow either ubiquitous or cell-specific expression of a reporter gene. Our study indicates the versatility of Tol2 for transient transgenesis, and provides a template for transgenesis work in other emerging reference species.</p></div

Metronidazole treatment does not affect general brain anatomy of <i>Platynereisdumerilii</i>.

<p>Confocal images of 10µm thick horizontal cryosections from premature adult transgenic <i>Platynereis</i> heads (r-opsin1::egfp-f2a-ntr), stained with antibodies against acetylated tubulin (A,B; green), and serotonin (C,D; red), as well as with the DNA-binding agent DAPI (E,F; blue). Panels G and H are merged views of all three channels. Animals (45-55 segments) were treated with DMSO (A,C,E,G) or 25mM metronidazole (B,D,F,H) for ~72-80hrs. No morphological differences were observed in the overall neuropil structure and wiring (B,H, compare to A, G) and the position and wiring of serotonergic neurons (D,H, compare to C, G) in ablated animals compared to controls. asterisks: center of neuropil, arrows: posterior serotonergic neuron cell bodies, yellow arrowheads: antennal nerve, ae: position of adult eyes; white arrowheads: position of GFP+ PRCs in control animals (A,G) and equivalent position in metronidazole-treated animals (B,H) Note absence of eGFP fluorescence in B,H, compared to A ,G, indicative of the successful ablation of the <i>r-opsin1+</i> PRCs. Scale bar: 50µm; dorsal views, anterior up. Also compare to reference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075811#B2" target="_blank">2</a>] for details of the brain scaffold.</p

Survival and transmission rates for the injection of Mos1-based fluorescent reporter constructs.

<p>Data are shown for both pMos{tuba::egfp}^frkt707 and pMos{r-opsin::egfp}^frkt890 injections. Injected zygotes (left column) were raised to mature animals (G₀; middle column), and EGFP expression in the offspring (G₁; right column) was monitored. Survival rate and transgenic founder rate are given as percentage of injected animals and mature animals, respectively.</p

Metronidazole induces apoptosis in transgenically labeled cells.

<p>(A-L) Head sections of mtz treated (A-D), DMSO treated (E-H) and untreated (I-L) premature adult <i>r-opsin1::eGFP-f2A-ntr</i> worms (eye PRCs and projections, green) processed for terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labelling (TUNEL) detection (red). (A-D) Apoptosis was detected in PRCs exposed to 25mM mtz after 28 hrs incubation, whereas transgenic animals treated with DMSO alone (E-H) or transgenic untreated animals (I-L), did not show staining above background. ae: adult eye. Scale bars C: 50µm; D: 15µm.</p