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First published online 11 February 2004
doi: 10.1242/dev.01004

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Overlapping mechanisms function to establish transcriptional quiescence in the embryonic Drosophila germline

Department of Molecular Biology, Princeton University, Princeton, NJ 0854, USA

View larger version (98K): [in a new window]   Fig. 1. Ectopic Bcd induces hb transcription in pole cells. Co-immunostaining of embryos with anti-ß-galactosidase (red) and anti-Vasa (green) antibodies. The anti-Vasa antibody is used to mark pole cells. (A,C,E) Anti-ß-galactosidase staining alone. (B,D,F) Merged images of anti-ß-galactosidase and anti-Vasa staining. (A,B) Embryos produced by wild-type females mated to males carrying the hb:lacZ reporter transgene. ß-galactosidase can be detected in somatic cells of wild-type blastoderm embryos in an anterior cap (not shown) and a posterior stripe (asterisk at posterior boundary); however, there is no evidence of ß-galactosidase expression in the pole cells. (C,D) Embryos produced by bcd nos3'UTR females mated with males carrying the hb:lacZ reporter transgene. Anti-ß-galactosidase staining reveals expression of the hb:lacZ reporter in pole cells of bcd-nos3'UTR embryos (yellow in D). As is evident here, expression of hb:lacZ is also detected in somatic cells at the posterior in some bcd-nos3'UTR embryos. In this respect, the hb:lacZ transgene differs from the endogenous hb gene as little if any Hb protein is detected in the posterior soma of bcd-nos3'UTR embryos. It seems likely that we are able to detect a low level of ß-galactosidase because this protein is much more stable than Hb. (E,F) Embryos produced by tsl mutant, bcd-nos3'UTR females mated with males carrying the hb:lacZ transgene. ß-Galactosidase is no longer detected in pole cells in the absence of tsl function. Indicative of the tsl mutation, the posterior boundary of the posterior hb stripe (asterisk) shifts anteriorly.

View larger version (74K): [in a new window]   Fig. 2. The level of CTD phosphorylation is altered in pole cells of bcd-nos3'UTR embryos. Co-immunostaining of embryos with monoclonal antibody H5 (green) and anti-Vasa antiserum (red). The H5 antibody specifically recognizes RNA polymerase II CTD repeats phosphorylated on serine 2. (A) Wild-type embryo. H5 antibody staining is readily apparent in the transcriptionally active somatic nuclei but not in the transcriptionally quiescent pole cells. (B) bcd-nos3'UTR embryo. H5 antibody stains pole cell nuclei in addition to the adjacent somatic nuclei, indicating that the level of CTD phosphorylation is elevated in these pole cells. (C) pgc embryo. H5 antibody stains pole cell nuclei in early blastoderm embryos. Note that all the somatic nuclei have not yet reached the periphery.

View larger version (127K): [in a new window]   Fig. 3. Germ cell migration defects in bcd-nos3'UTR embryos. Immunostaining of stage 13 embryos with anti-Vasa antibody. (A) Wild-type embryo. Germ cells have coalesced into the somatic gonad. (B) bcd-nos3'UTR embryo. Many germ cells are found scattered over several segments.

View larger version (50K): [in a new window]   Fig. 4. Ectopic expression of bcd at the posterior pole induces Sxl expression. Merged images of embryos co-immunostained with anti-ß-galactosidase (red) and anti-Vasa (green) antibodies. (A) Embryo produced by wild-type female mated to male carrying the Sxl-Pe:lacZ reporter transgene. ß-galactosidase is detected in somatic cells but not in pole cells. (B) Embryo produced by bcd-nos3'UTR female mated to male carrying the Sxl-Pe:lacZ reporter transgene. ß-galactosidase is detected in both somatic and pole cells. (C) Embryo produced by bcd-nos3'UTR female mutant for tsl mated to male carrying the Sxl-Pe:lacZ reporter transgene. Expression of the Sxl-Pe:lacZ reporter is no longer detected in pole cells when tsl function is removed. Embryos shown in A-C are judged to be female by the activation of Sxl-Pe in somatic cells.

View larger version (71K): [in a new window]   Fig. 5. torso-nos3'UTR can activate transcription of tailless-lacZ in early pole cells. Wild-type or torso-nos3'UTR females were mated to males carrying the tll:lacZ transgene. Embryos were co-immunostained with anti-Vasa (green) and anti-ß-galactosidase antibodies (red). All images show embryos at cycle 10. Wild-type embryo labeled with (A) both the antibodies or with (A') ß-galactosidase antibody. torso-nos3'UTR embryo labeled with (B) both antibodies or (B') only with ß-galactosidase antibody. Little or no ß-galactosidase is observed in the wild-type embryo, while a low but clearly detectable level of ß-galactosidase is evident in the torso-nos3'UTR embryos.

View larger version (71K): [in a new window]   Fig. 6. tailless-lacZ reporter is turned on in the newly formed pole cells of embryos maternally compromised for pgc activity. Wild-type females or females carrying two copies of the antisense pgc transgene were mated to males carrying the tll-lacZ reporter. The two panels at the top show pgc embryos probed with ß-galactosidase antibody and visualized by DAB staining. The panel on the left shows a cycle 10 embryo which has a higher number than normal pole cells. Several of these pgc pole cells express high levels of ß-galactosidase, while others have little if any ß-galactosidase. At this stage, wild-type tll-lacZ embryos probed with ß-galactosidase antibody show no detectable DAB staining in either the pole cells or the soma. The panel on the right shows a cycle 10 pgc embryo that has ß-galactosidase positive `Pole cells' at unusual positions (marked with arrows). Some of these are in the interior of the embryo, while others are near the surface. The pole cells shown in the middle and lower panels were co-immunostained with anti-Vasa (green) and anti-ß-galactosidase antibodies (red). All images show embryos at cycle 10. Note that some of the `pole cells' in the pgc embryo in the bottom panels express very high levels of ß-galactosidase but have little or no Vasa protein. Note also that some of the Vasa-positive pole cells are in the interior of the embryo rather than at the surface at the posterior pole.

View larger version (82K): [in a new window]   Fig. 7. MAP kinase signaling is elevated in the pole cells compromised for pgc function. (A,B) Wild-type embryos were stained with anti ERK antibodies and signal was visualized with DAB. (A) A syncytial blastoderm embryo displays increased levels of staining at both the termini. Very little, if any, signal is detected in the pole cells. (B) Signal specific for anti-ERK antibodies completely disappears by cellular blastoderm stage. (C,D) Pre-syncytial cycle 10 embryos co-immunostained with monoclonal anti-ERK antibody (green) and anti-Vasa antiserum (red). (C',D') The same embryos as in C and D labeled with only anti-ERK antibody. Pole cells from the wild-type embryo (C and C') have only a low level of anti-ERK specific signal whereas pole cells compromised for pgc function (D,D') have much higher levels.

View larger version (136K): [in a new window]   Fig. 8. H3meK4 is absent in newly formed wild-type Drosophila pole cells but is present in pole cells depleted for pgc function. Wild-type embryos (0-3 hours) and pgc- embryos (0-3 hours) were fixed and co-immunostained with anti-H3meK4 (red) and anti Vasa (green) antibodies. Nuclear density was estimated using a DNA dye, Hoechst (imaged in blue as seen in A,B). (A) Pre-syncytial wild-type blastoderm embryo probed with both antibodies and Hoechst dye. (A') Same embryo showing very little H3meK4-specific signal either in the soma or in the newly formed pole cells. (B) Similar age embryo compromised for pgc function probed with both antibodies and Hoechst dye showing the presence of K4 signal in the newly formed pole cells. (B') Same embryo. Pole cells stained with H3meK4 specific antibodies marked with an arrow and an arrowhead. (C) Wild-type syncytial blastoderm embryo probed with both antibodies. H3meK4 specific signal appears in the soma but pole cells are still devoid of the signal. (C') Same embryo showing just the H3MeK4 staining. (D) A late syncytial blastoderm embryo compromised for pgc function probed with both the antibodies. H3MeK4 specific staining is reduced in almost all pole cells, except in two slightly internally positioned Vasa-positive cells that still show considerable level of signal (marked with an arrow and an arrowhead). (D') Same embryo showing just H3MeK4-specific signal.