A functional antagonism between the pgc germline repressor and torso in the development of somatic cells

Abstract

Segregation of the germline is a fundamental event during early development. In Drosophila, germ cells are specified at the posterior pole of the embryo by the germplasm. As zygotic expression is activated, germ cells remain transcriptionally silent owing to the polar granule component (Pgc), a small peptide present in germ cells. Somatic cells at both the embryonic ends are specified by the torso (Tor) receptor tyrosine kinase, and in tor mutants the somatic cells closer to the germ cells fail to cellularize correctly. Here, we show that extra wild-type gene copies of pgc cause a similar cellularization phenotype, and that both excessive pgc and a lack of tor are associated with an impairment of transcription in somatic cells. Moreover, a lack of pgc partly ameliorates the cellularization defect of tor mutants, thus revealing a functional antagonism between pgc and tor in the specification of germline and somatic properties. As transcriptional quiescence is a general feature of germ cells, similar mechanisms might operate in many organisms to 'protect' somatic cells that adjoin germ cells from inappropriately succumbing to such quiescence.

Figure 1

The pole–hole phenotype of tor and 6x[pgc] embryos, and suppression of the tor^– pole–hole phenotype by pgc and vas. Posterior poles of wild type (A,F,K), tor^– (B,G,L), 6x[pgc] (C,H,M) tor^–,pgc^– (D,I,N) and vas^–,tor^– embryos (E,J,O). Anti-neurotactin (Nrt; red) labels somatic but not germ cells; DAPI labels nuclei (blue); and in anti-Vas labels germ cells (green). Groups of nuclei fall into the yolk in tor and 6x[pgc] mutants (arrows in G and H), and some cells fail to complete cellularization as shown by the lack of a basal membrane (see arrowhead in M) and many cells have lost the typical epithelial elongated shape. In (L) tor and (M) 6x[pgc] embryos, germ cells are found in the ‘hole' between the somatic cells. The pole–hole phenotype is partly suppressed in tor,pgc double mutants, whereas somatic nuclei still fall into the yolk (I), somatic cells are better arranged (D) and germ cells remain closer to the periphery (N). Suppression of the pole–hole phenotype is complete in vas,tor mutants (E,J,O); note that germ cells are not formed in these embryos because of the vas mutation (O). DAPI, 4′,6-diamidino-2-phenylindole; pgc, polar granule component; tor, torso; vas, vasa; wt, wild type.

Figure 2

Frames of time-lapse movies showing the development of wild-type, tor and 6x[pgc] embryos. Posterior embryonic poles of wild-type, tor and 6x[pgc] embryos with a His2avGFP transgene to label nuclei. Groups of nuclei from tor and 6x[pgc] embryos fall into the yolk at various time points during cellularization at nuclear cycle 14 (arrowheads). Occasionally, a few nuclei fall into the yolk in the wild-type embryo (arrows). Frames in the figure are separated by 12′30' intervals. GFP, green fluorescent protein; pgc, polar granule component; tor, torso; wt, wild type.

Figure 3

The ectopic expression of tll in the germ cells of pgc mutants is independent of tor activity. Posterior pole of cellular blastoderms hybridized with a tll probe. (A) tll is excluded from germ cells of wild-type embryos. (B) In pgc mutants, tll is found in the germ cells. (C) In tor mutants, tll is absent from the posterior cells. (D) In tor,pgc double mutants, tll is absent from the posterior somatic cells but is expressed in the germ cells. pgc, polar granule component; tll, tailless; tor, torso; wt, wild type.

Figure 4

Transcriptional impairment associated with the pole–hole phenotype. (A,C,E,G) Posterior pole of cellular blastoderm labelled with an antibody that recognizes the active form of the RNA polymerase type II (pSer; green) and the germline specific Vas antibody (blue), and graphical representations of pSer levels in the posterior somatic nuclei (for exact numbers and quantification see the supplementary information online). Levels of pSer antibody staining are lower in the somatic cells next to the germ cells in tor (C) and 6x[pgc] (E), but similar to those in the other somatic cells in tor,pgc double mutants (G). (B,D,F,H) Posterior pole of blastoderms hybridized with a slam probe. A decrease in the signal can be detected in the somatic cells closer to the germ cells in tor (D) and 6x[pgc] (F) but not in tor,pgc double mutants (H). In (H), slam transcripts in the germ cells are due to the pgc mutation. pgc, polar granule component; slam, slow as molasses; tor, torso; wt, wild type.

Figure 5

pgc accumulates at the germ cells and surrounding cytoplasm. Posterior pole of a wild-type embryo hybridized with a pgc probe (A), stained with Pgc (B), and Vas antibodies (C), withWGA that labels the membranes (D) and a merge of WGA (blue) and anti-Vas (green) staining (E). pgc RNA accumulates in the germ cells and can also be detected in the surrounding somatic cytoplasm (arrows and inset with magnification). Similarly, Pgc protein accumulates in the germ cells and some traces can also be detected in cells that do not show staining for the germ-specific Vas protein (arrowheads and insets for more detail). We detected pgc RNA in the somatic cytoplasm surrounding the germ cells in 87% of wild-type embryos (n=30) and in 100% of 6x[pgc] embryos (n=42), and traces of Pgc protein in 30% of wild-type embryos (n=27) and 58% of 6x[pgc] embryos (n=24). (F) In germ cells, pgc represses gene transcription inhibiting the somatic fate and allowing the correct segregation of the germline. Other germplasm components probably participate in this process. Germ-cell fate is impaired by the overexpression of the Tor receptor (Martinho et al 2004). In the posterior somatic cells, the Tor pathway triggers the expression of the genes responsible for terminal fate and, in addition, counteracts the deleterious effect of pgc and other germplasm components in the somatic cellularization (asterisk). pgc, polar granule component; Tor, torso; Vas, vaso; WGA, wheat germ agglutinin; wt, wild type.