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First published online 6 July 2005
doi: 10.1242/dev.01921

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Dose-dependent control of proliferation and sperm specification by FOG-1/CPEB

¹ Cellular and Molecular Biology Training Program, University of Wisconsin-Madison, Madison, WI 53706, USA
² Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA
³ Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI 53706, USA

View larger version (69K): [in a new window]   Fig. 1. FBF and FOG-1 control larval germline proliferation. (A-C,F) DAPI-stained germlines. Broken lines indicate germlines; small arrowheads indicate sperm; large arrowheads indicate oocytes or oocyte-like cells; arrow in A-E indicates distal end; vul, vulva. fog-1, fog-1(q250). (A) fog-1 homozygote. Size of adult germline is normal, but only oocytes are made (Barton and Kimble, 1990). (B) fbf-1 fbf-2 adult. Germline is reduced to 60 germ cells/arm with only sperm (Crittenden et al., 2002). (C) fog-1; fbf-1 fbf-2 triple mutant. Germline is reduced to 10 germ cells (white arrowheads) and no sperm are made. (D) fog-1; fbf-1 fbf-2 triple mutant, stained with antibodies to the germ cell marker PGL-1; few germ cells are present (white arrowheads). (E) fog-1; fbf-1 fbf-2 Nomarski micrograph; few germ cells are present (white arrowheads). (F) L2 fog-1; fbf-1 fbf-2. Arrow indicates crescent-shaped nucleus typical of early meiosis. Scale bars: 10 µm. (G) Larval germline proliferation. eL1, early L1; lL1, late L1. n4 at all stages.

View larger version (69K): [in a new window]   Fig. 2. FOG-1 dose affects germline proliferation. (A) The FOG-1 protein. Red indicates region used to generate FOG-1 antibodies (Ab, inverted Y); black boxes indicate RRM motifs; gray boxes indicate C/H Zn-finger motif. Sites of fog-1 mutations are marked and bracketed by class of molecular lesion. (B-D) Broken lines indicate germlines; small arrowheads indicate sperm; large arrowheads indicate oocytes or oocyte-like cells; arrow indicates distal end; vul indicates vulva. (B) fog-1(q250)/+; fbf-1 fbf-2 Nomarski micrograph. fog-1/+; fbf-1 fbf-2 animals generate more germ cells than fbf-1 fbf-2 animals; only sperm are made. (C) fog-1(q250)/+; fbf-1 fbf-2, DAPI stained. (D) fog-1(q325); fbf-1 fbf-2 Nomarski micrograph. fog-1(q325); fbf-1 fbf-2 animals generate more germ cells than fbf-1 fbf-2 animals, but fog-1(q325) homozygotes only make oocytes. Scale bars: 10 µm.

View larger version (89K): [in a new window]   Fig. 3. FBF and FOG-1 act downstream of Notch signaling. (A-C) Large arrowheads, oocytes or oocyte-like cells; arrow indicates distal end; vul, vulva. (A) glp-1(gf); fog-1(RNAi); fbf(RNAi) germlines possess small granular oocyte-like cells extending to distal end (arrow). (B) glp-1(gf); fbf(RNAi) germlines are tumorous. (C) glp-1(gf); fog-1(RNAi) germlines are tumorous. Scale bars: 10 µm. (D) Model for FBF and FOG-1 in germline proliferation pathway.

View larger version (68K): [in a new window]   Fig. 4. FBF acts upstream of fog-1 in the sperm/oocyte decision. (A-D) Broken lines indicate germlines. Green indicates sperm-specific SP56 marker; red indicates oocyte-specific RME-2 marker. Genotypes, stages and markers are indicated in micrographs. (A,C) fbf-1 fbf-2 mutants express sperm marker, but not oocyte marker. (B,D) fog-1; fbf-1 fbf-2 mutants do not express sperm, but express oocyte marker. Scale bars: 10 µm. (E) Model for FBF repression of multiple genes in germline sex determination pathway.

View larger version (66K): [in a new window]   Fig. 5. FBF binds specifically to elements in fog-1 and fog-3 3'UTRs. (A) Putative FBF binding elements (FBEs) in fog and fem 3'UTRs; black triangles, elements that bind in vitro; gray triangles, elements that do not bind in vitro. (B) Nucleotide sequence of predicted FBF binding elements. Sequences are aligned by conserved UGU motif (bold). Mutated nucleotides are lowercase. [UUUUU towards 3' end of fog-1 FBE bc RNA was disrupted to increase expression in three-hybrid system (indicated by `a').] (C) Yeast three-hybrid assay. (D) Yeast three-hybrid results. Error bars represent standard deviation of at least four repetitions. gld-1 FBEa, element in gld-1 3'UTR (Bernstein et al., 2005; Crittenden et al., 2002); fem-3 PME, element in fem-3 3'UTR (Zhang et al., 1997), which is called fem-3 FBEa in A. (E) FBF-2 binds FBEs in fog-1 and fog-3 3'UTRs. Protein concentrations were 0.5 nM, 1 nM and 5 nM, then consecutively doubled to reach 640 nM. Apparent binding constants are shown below each shift.

View larger version (58K): [in a new window]   Fig. 6. FBF represses fog-1 expression in vivo. (A-J) Arrow indicates distal end. Scale bars: 10 µm. (A-E,G-J) Animals or dissected germlines stained with -FOG-1 antibodies. Broken lines indicate germlines in A-D. (A) Wild-type L2. FOG-1 is barely detectable. (B) Wild-type L3. FOG-1 is abundant in proximal germline where cells are destined to become sperm; FOG-1 is low in distal germline where cells remain in mitotic cell cycle. (C,D) L2 fbf-1 fbf-2. FOG-1 is more abundant than in wild type (compare with A) and its distribution is not graded. (D) L3 fbf-1 fbf-2. FOG-1 is more abundant than in wild type (compare with B) and its distribution is not graded. (E) Wild-type adult male germline. MR, mitotic region; TZ, transition zone. FOG-1 levels are low in mitotic region and become high as germ cells begin entry into meiosis in proximal part of mitotic region and transition zone. FOG-1 disappears in more mature stages of meiotic prophase. (F) Wild-type adult male germline hybridized with fog-1 antisense probe. fog-1 mRNA has same pattern as FOG-1 protein (compare with E). Sense probe has no detectable staining (not shown). (G) fog-1(q250) homozygous male germline. No FOG-1 protein is detectable. (H) Wild-type adult hermaphrodite germline. No FOG-1 protein is detectable. (I) Adult gld-3 nos-3 tumorous germline. FOG-1 levels are low. (J) Adult fbf-1 fbf-2 gld-3 nos-3 tumorous germline. FOG-1 levels increase in the absence of FBF (compare with I).

View larger version (34K): [in a new window]   Fig. 7. Model for control of germline fate by FOG-1 abundance. (A) Threshold model for FOG-1 control of two germline fates. [FOG-1], hypothetical FOG-1 concentration; broken line, threshold of FOG-1 concentration; yellow, low FOG-1 promotes mitosis; blue, high FOG-1 that specifies spermatogenesis. x-axis, developmental time. In fbf-1 fbf-2 double mutants (blue line), FOG-1 protein increases with time and ultimately promotes spermatogenesis in all germ cells. In fog-1/+; fbf-1 fbf-2 animals (green line), FOG-1 levels are lower than in fbf-1 fbf-2 double mutants, but they still increase with time; as a result, mitosis continues longer and more germ cells are generated before FOG-1 accumulates to a level that promotes the sperm fate. In fog-1; fbf-1 fbf-2 triple mutants (red line), no FOG-1 is generated, few mitotic divisions occur (presumably under control of some other regulator), and no sperm are specified. The FOG-1 increase with time is depicted as linear for simplicity, but it may well increase non-linearly because of a combination of FBF repression and positive autoregulation (see text). (B) Model for FOG-1 spatial gradient in adult male germline. FOG-1 levels are depicted by font size. We suggest that low FOG-1 in the mitotic region promotes proliferation, whereas high FOG-1 in distal MR and transition zone specifies the sperm fate as germ cells enter meiosis.