Mechanisms and protective strategies for Neriifolin-induced DA neuronal death.

<p>(A) Treatment with Neriifolin in the presence of high K⁺ in the medium significantly decreased DA neuronal loss compared to the treatment with Neriifolin alone. Data are the averages ± SEM from 6 embryos in a single experiment that was repeated twice with similar results. (B) Treatment with Neriifolin in the presence of low Na⁺ in the medium significantly decreased DA neuronal loss compared to the treatment with Neriifolin alone. Data are the averages ± SEM from 8 embryos in a single experiment that was repeated twice with similar results. (C–D) Treatment with either Quercetin (C) or Ascorbic Acid (D) significantly decreased DA neuronal death compared to treatment with Neriifolin alone. Data are the averages ± SEM from 8 embryos in a single experiment that was repeated twice with similar results.</p

Neriifolin selectively impairs the survival of mammalian DA neurons derived from embryonic stem cells (ESCs).

<p>(A) Schematic diagram of the protocol used to induce DA neuron differentiation from mouse ESCs. (B) Representative views from wells with different treatment, showing the percentage of TH⁺ neurons (TH, a marker of DA neurons, in red) or all neurons (NeuN, a neuronal marker, in green) among total cells (DAPI). Compared to DMSO control (upper panel), those cells treated with Neriifolin from Day 7 to 11 (middle panels) showed no decrease in the percentage of TH⁺ neurons and all neurons; but those cells treated with Neriifolin from day 11 to 14 (lower panels) showed significant less percentage of DA neurons. (C) Quantification of the percentage of TH⁺ neurons among total cells. Fold change of treatment vs DMSO control was shown. (D) Quantification of fold change of TH⁺ neurons and all neurons. In both (C) and (D), data are the averages ± SEM of triplicates from a single experiment that was repeated 3 times with similar results.</p

Human <i>atp1a3</i> rescues DA neurons in Neriifolin-treated embryos.

<p>(A) The expression pattern of <i>atp1a3a</i> in wild-type embryos at 48 hpf. (B) The schematic diagram of the plasmid constructs used for the rescue experiments in zebrafish embryos. (C) RT-PCR detection of the expression of human <i>atp1a3</i> in zebrafish embryos after injection and heat shock. (D–E) Quantification (D) and representative images (E) of VFB DA neurons in 5 µM Neriifolin-treated embryos that express either GFP or human <i>atp1a3</i>. Data are the averages ± SEM from 9 embryos in a single experiment that was repeated twice with similar results.</p

Neriifolin-induced DA neuronal death is apoptotic and requires p53.

<p>(A–D′″) Low (A–D) and high (A′–D′″) magnification views of VFB DA neurons in control (A-A′″) vs Neriifolin-treated embryos (B-B′″), and sympathetic (Sym) NA neurons in control (C-C′″) vs Neriifolin-treated embryos (D-D′″). Ventral views of 60 hpf embryos were shown. Neriifolin treatment was carried out from 24 hpf to 60 hpf. (E) Injection of the p53-MO into embryos at one-cell stage protected DA neurons from cell death induced by Neriifolin. Data are the averages ± SEM from 6 embryos in a single experiment that was repeated twice with similar results.</p

Germ cells from <i>tkv</i> germline clone mothers have spectrosome defects. Panels A–C: Stage 15 <i>tkvm-z+</i> gonads stained for Vasa (green) and Spectrin (red).

<p><b>Panels D–F:</b> Same embryos as in A–C, except that a gray scale is used to image Spectrin. <b>A, D.</b> Germ cells exhibiting normal spectrosome morphology. Spectrosomes are spherical, well- defined and, show a strong Spectrin signal. <b>B, E.</b> Germ cells showing weak, diffuse Spectrin staining or irregularly shaped spectrosomes (arrows). <b>C, F.</b> Germ cells with fragmented (arrows) or missing (arrowhead) spectrosome.</p

BMP Signaling and the Maintenance of Primordial Germ Cell Identity in Drosophila Embryos

<div><p>The specification of primordial germ cells (PGCs) and subsequent maintenance of germ-line identity in <i>Drosophila</i> embryos has long been thought to occur solely under the control of cell-autonomous factors deposited in the posterior pole plasm during oogenesis. However, here we document a novel role for somatic BMP signaling in the maintenance of PGC fate during the period leading up to embryonic gonad coalescence. We find that PGCs fail to maintain their germline identity when BMP signaling is compromised. They initiate but are unable to properly assemble the germline stem cell-specific organelle, the spectrosome, and they lose expression of the germline-specific gene Vasa. BMP signaling must, however, be finely tuned as there are deleterious consequences to PGCs when the pathway is excessively active. We show that one mechanism used to calibrate the effects of BMP signals is dependent on the Ubc9 homolog Lesswright (Lwr).</p></div

Effects of excess of BMP signaling on Vasa protein.

<p><b>A.</b> Stage 15 wild type embryo. Gonad is tightly coalesced and all germ cells stain strongly for Vasa. <b>B.</b> Stage 15 <i>nos-Gal4/UAS-dpp</i> embryo. Most germ cells stain in this embryo only faintly for Vasa. <b>C.</b> Blow-up grey scale image of Vasa accumulation in the PGCs shown in the embryos in panels A and B. Upper panel: WT PGCs. Lower panel: <i>nos-Gal4/UAS-dpp</i> PGCs. <b>D.</b> Vasa expression in WT, <i>nos-Gal4/UAS-dpp</i> and <i>nos-Gal4/UAS-lwrDN</i> stage 15 embryos. Embryos have a single copy of both the <i>Gal4</i> driver and the <i>UAS</i> transgene as indicated. Dark blue: Percent of PGCs having normal levels of Vasa. Light blue: Percent of PGCs having an obvious reduction in the levels of Vasa compared to their sibs in the same embryo. For this analysis, Vasa levels were scored by comparing staining in the PGCs of the same embryo, not between different embryos. In a wild type embryo (processed in parallel) all PGCs have a similar and uniformly high level of Vasa protein. In contrast, when BMP signaling is upregulated, the level of Vasa protein in different PGCs in the same embryo can differ.</p

Vasa protein is lost when the ability of PGCs to respond to BMP signals is compromised.

<p><b>A.</b> Stage 13 <i>nos-Gal4;UAS-dsmurf</i> embryo. Germ cells show unequal Vasa accumulation. <b>B.</b> Stage 15 <i>nos-Gal4;UAS-dsmurf</i> embryo. Again, germ cells show uneven Vasa accumulation. <b>C.</b> A substantial percentage of germ cells receiving insufficient BMP signaling display reduced Vasa accumulation. Dark blue: Percentage of germ cells having high levels of Vasa. Light blue: Percentage of germ cells having reduced levels of Vasa. For this analysis, Vasa levels were scored by comparing staining in the PGCs of the same embryo, not between different embryos. In a wild type embryo (processed in parallel) the PGCs have a similar and uniformly high level of Vasa protein. In contrast, when BMP signaling is compromised, the level of Vasa protein in different PGCs in the same embryo differs. In addition to the variability in Vasa protein levels shown here, a comparison with wild type embryos processed in parallel using the confocal microscope indicates that Vasa protein levels are generally reduced in PGCs compared to wild type when BMP signaling is compromised. Again, as was the case for germ cell loss when Dpp is over expressed, the reduction in Vasa levels does not appear to be sex-specific.</p

Mid-stage PGCs can respond to Dpp signaling.

<p><b>A, B.</b> Stage 12 wild type embryo. <b>C, D.</b> Stage 12 <i>twi-Gal4/+;UAS-dpp/+</i> embryo. Panels A and C: Vasa imaged in red; pMad imaged in green. Panels B and D: gray scale image of pMad. In wild type (A, B) nuclear pMad levels are near background; however there does seem to be some cytoplasmic protein in PGCs (seen as a ring around each PGC nucleus: see arrow in panel B). In <i>twi-Gal4/+;UAS-dpp/+</i> embryos nuclear pMad is readily detected (see panel D). Dpp expressing embryos were generated by mating virgin females carrying two copies of the <i>twi-Gal4</i> driver with males carrying two copies of <i>UAS-dpp.</i></p

pMad is enriched in early pole cell nuclei.

<p><b>A, B.</b> Stage 5 wild type blastoderm stage embryo shows pole cells (with nuclear accumulation of pMad. Dorsal somatic nuclei also show high levels of nuclear pMad. <b>C, D.</b> Stage 6 wild type embryo also shows high nuclear pMad levels in the pole cells and dorsal somatic cells. Panels A and C: Vasa imaged in red; pMad imaged in green. Panels B and D: gray scale image of pMad.</p