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

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

<i>lesswright</i> downregulates BMP signaling in PGCs.

<p>Panels <b>A, B, C:</b> Stage 15 embryos. <b>A:</b> Wild type. <b>B: </b><i>nos-Gal4/UAS-dpp</i> embryo showing reduction in PGCs. <b>C: </b><i>UAS-lwr/+;nos-Gal4/UAS-dpp</i> embryo showing intermediate number of PGCs indicating partial rescue of PGC loss phenotype observed in <i>nos-Gal4/UAS-dpp</i> embryos. <i>UAS-lwr/+; nos-Gal4/UAS-dpp</i> stage 15 embryos have an average of 8 PGCs per gonad as compared to 5 PGCs/gonad for <i>nos-Gal4/UAS-dpp</i> and 12 PGCs/gonad for wild type. All transgenic embryos had a single copy of the <i>Gal4</i> driver.</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

Overexpression of Dpp has deleterious effects on PGCs.

<p><b>A, C, E.</b> Stage 13. <b>B, D, F.</b> Stage 15. <b>A, B.</b> Wild type embryos. <b>C, D.. </b><i>twi-Gal4/+; UAS-dpp/+</i> embryos. Germ cell migration defects are evident in the stage 13 embryo (C) while the gonad of the stage 15 embryo (D) has a reduced number of PGCs. Patterning defects are evident in both. <b>E, F.. </b><i>nos-Gal4/UAS-dpp</i> embryos. Both stage 13 (E) and 15 (F) embryos have fewer than normal PGCs. Only a minor germ cell migration defect is evident in (E). There are no obvious patterning defects. <b>G.</b> The number of germ cells per gonad is reduced in embryos overexpressing Dpp in the mesoderm (twi-Gal4/UAS-dpp) or in the gonad (nos-Gal4/UAS-dpp) as compared to wild type. The deleterious effects of <i>dpp</i> over-expression did not appear to be sex specific as both male and female embryo showed germ cell loss (data not shown). Embryos have a single copy of both the <i>Gal4</i> driver as indicated and the <i>UAS-dpp</i> transgene.</p

<i>dpp</i> is needed to maintain spectrosome integrity and PGC identity Panels A, C, E, and G: Vasa image in red, Spectrin imaged in green.

<p><b>Panels B, D, F, and H:</b> Grey scale images of Spectrin. <b>A, B.</b> PGCs in stage 15 <i>dpp−/CyO,P</i> gonads have wild type spectrosomes and normal levels of Vasa protein. The spectrosomes of stage 15–16 gonads are large well-defined spherical structures that contain Spectrin (green/grey) and, Hts (not shown). The PGCs have a spherical shape and contain high levels of cytoplasmic Vasa (red). <b>C–H. </b><i>dpp−/−</i> embryos. <b>C, D.</b> One germ cell shows normal spectrosome morphology (arrow), while others have a diffusely labeled spectrosome (arrowhead) or lack any visible spectrosome (*). Several germ cells also show abnormal morphology. <b>E, F.</b> A cluster of germ cells. Some have normal looking spectrosome foci, while others appear to have extra spectrosome foci (arrow in F). <b>G, H.</b> One germ cell shows a complete loss of Vasa but has retained some Spectrin (arrowhead in G) while others show fragmented spectrosomes (arrow in H).</p

pMad accumulation in stage 15 PGCs.

<p><b>A–C.</b> Stage 15 embryos stained for Vasa (red) and pMad (green). <b>D–F.</b> Grey scale images of the same embryos showing only pMad. A,C: Wild type gonad. B,E: <i>nos- Gal4/UAS-dpp</i> PGCs. Levels of nuclear and cytoplasmic pMad increase in PGS when Dpp is overexpressed. C,F: <i>nos-Gal4/UAS-lwrDN</i> PGCs. Levels of nuclear and cytoplasmic pMad increase when LW activity is inhibited. All embryos carry a single copy of the driver and a single copy of the <i>UAS</i> transgene.</p