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C. elegans FOG-3/Tob can either promote or inhibit germline proliferation, depending on gene dosage and genetic context.

Snow JJ, Lee MH, Verheyden J, Kroll-Conner PL, Kimble J - Oncogene (2012)

Bottom Line: Thus, fog-3 mutants possess fewer germ cells than normal, a modest but reproducible decrease observed for each of two distinct fog-3 alleles.Finally, we show that FOG-3 and FBF work together to promote tumor formation in animals carrying oncogenic Notch mutations.A similar effect was not observed when germline tumors were induced by manipulation of other regulators; therefore, this FOG-3 tumor-promoting effect is context dependent.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA.

ABSTRACT
Vertebrate Tob/BTG proteins inhibit cell proliferation when overexpressed in tissue-culture cells, and they can function as tumor suppressors in mice. The single Caenorhabditis elegans Tob/BTG ortholog, FOG-3, by contrast, was identified from its loss-of-function phenotype as a regulator of sperm fate specification. Here we report that FOG-3 also regulates proliferation in the germline tissue. We first demonstrate that FOG-3 is a positive regulator of germline proliferation. Thus, fog-3 mutants possess fewer germ cells than normal, a modest but reproducible decrease observed for each of two distinct fog-3 alleles. A similar decrease also occurred in fog-3/+ heterozygotes, again for both fog-3 alleles, revealing a haplo-insufficient effect on proliferation. Therefore, FOG-3 normally promotes proliferation, and two copies of the fog-3 gene are required for this function. We next overexpressed FOG-3 by removal of FBF, the collective term for FBF-1 and FBF-2, two nearly identical PUF RNA-binding proteins. We find that overexpressed FOG-3 blocks proliferation in fbf-1 fbf-2 mutants; whereas germ cells stop dividing and instead differentiate in fbf-1 fbf-2 double mutants, they continue to proliferate in fog-3; fbf-1 fbf-2 triple mutants. Therefore, like its vertebrate Tob/BTG cousins, overexpressed FOG-3 is 'antiproliferative'. Indeed, some fog-3; fbf-1 fbf-2 mutants possess small tumors, suggesting that FOG-3 can act as a tumor suppressor. Finally, we show that FOG-3 and FBF work together to promote tumor formation in animals carrying oncogenic Notch mutations. A similar effect was not observed when germline tumors were induced by manipulation of other regulators; therefore, this FOG-3 tumor-promoting effect is context dependent. We conclude that FOG-3 can either promote or inhibit proliferation in a manner that is sensitive to both genetic context and gene dosage. The discovery of these FOG-3 effects on proliferation has implications for our understanding of vertebrate Tob/BTG proteins and their influence on normal development and tumorigenesis.

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Overexpressed FOG-3 blocks proliferation in fbf-1 fbf-2 mutants. (a) Germlines were scored for presence of undifferentiated cells in the distal gonad (Distal undiff) and for gamete formation in adults (Proximal germline); Mog (masculinization of germline), sperm only; Fog, oocytes only; Tum, undifferentiated throughout germline; other sterile. (b–g) Adult fog-3; fbf-1 fbf-2 gonads, all 1 day past mid-L4. Scale bars represent 50 μM. Gonads are outlined. Empty arrowhead marks distal end. (b) Nomarski micrograph of Fog germline with oocytes marked by solid arrowheads, shown in intact animal. The distal germline has many undifferentiated cells. Germlines of this appearance could be fertile and make viable progeny when crossed to wild-type males, showing that FBF is not required for oogenesis or embryogenesis. (c) Hoechst-stained germline from the “other sterile” group. The germline has distal undifferentiated germ cells and proximal germ cells that had deteriorated (outlined by solid line). (d,e) Distal region of same Fog gonad contains undifferentiated cells with nuclear morphology characteristic of mitotic cell cycle. (d) DAPI-stained. (e) EdU incorporation highlights actively cycling cells. (f,g) Same gonad with a tumorous germline. (f) DAPI-stained. (g) Stained with anti-phosphorylated histone H3 to mark nuclei of cells in late G2 and M-phase (41).
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Figure 3: Overexpressed FOG-3 blocks proliferation in fbf-1 fbf-2 mutants. (a) Germlines were scored for presence of undifferentiated cells in the distal gonad (Distal undiff) and for gamete formation in adults (Proximal germline); Mog (masculinization of germline), sperm only; Fog, oocytes only; Tum, undifferentiated throughout germline; other sterile. (b–g) Adult fog-3; fbf-1 fbf-2 gonads, all 1 day past mid-L4. Scale bars represent 50 μM. Gonads are outlined. Empty arrowhead marks distal end. (b) Nomarski micrograph of Fog germline with oocytes marked by solid arrowheads, shown in intact animal. The distal germline has many undifferentiated cells. Germlines of this appearance could be fertile and make viable progeny when crossed to wild-type males, showing that FBF is not required for oogenesis or embryogenesis. (c) Hoechst-stained germline from the “other sterile” group. The germline has distal undifferentiated germ cells and proximal germ cells that had deteriorated (outlined by solid line). (d,e) Distal region of same Fog gonad contains undifferentiated cells with nuclear morphology characteristic of mitotic cell cycle. (d) DAPI-stained. (e) EdU incorporation highlights actively cycling cells. (f,g) Same gonad with a tumorous germline. (f) DAPI-stained. (g) Stained with anti-phosphorylated histone H3 to mark nuclei of cells in late G2 and M-phase (41).

Mentions: To assess the effect of overexpressed FOG-3 on germline proliferation, we compared fbf-1 fbf-2 mutant germlines with and without FOG-3. For these studies, we focused on adults, 24 hours past the L4 stage, and first asked whether germ cells were undifferentiated or differentiated in the distal gonad (Figure 3a, Distal undiff). Prior work showed that the distal gonads of wild-type animals and fog-3 mutants maintain undifferentiated germ cells throughout adulthood (15, 25, 26), but that the distal gonads of late L4 fbf-1 fbf-2 larvae contain no undifferentiated germ cells because all their germ cells differentiate into sperm (21). We first confirmed these conclusions for the distal germlines of wild-type, fog-3 and fbf-1 fbf-2 late L4 larvae (Figure 3a, lines 1–3). We then examined the effect of removing FOG-3 in fbf-1 fbf-2 mutants. In fog-3; fbf-1 fbf-2 triple mutant adults, the distal germ cells all appeared undifferentiated (Figure 3a, line 4; Figures 3b and c). A similar effect was seen when FOG-3 was depleted using RNAi (Figure 3a, line 6) or reduced by one gene copy (Figure 3a, line 7). To ask if these distal undifferentiated cells in fog-3; fbf-1 fbf-2 germlines were cycling, we treated animals with the thymidine analog EdU, an S-phase marker, and found that nuclei among the undifferentiated germ cells were labeled (Figures 3d and e). Therefore, these undifferentiated nuclei are progressing through the cell cycle.


C. elegans FOG-3/Tob can either promote or inhibit germline proliferation, depending on gene dosage and genetic context.

Snow JJ, Lee MH, Verheyden J, Kroll-Conner PL, Kimble J - Oncogene (2012)

Overexpressed FOG-3 blocks proliferation in fbf-1 fbf-2 mutants. (a) Germlines were scored for presence of undifferentiated cells in the distal gonad (Distal undiff) and for gamete formation in adults (Proximal germline); Mog (masculinization of germline), sperm only; Fog, oocytes only; Tum, undifferentiated throughout germline; other sterile. (b–g) Adult fog-3; fbf-1 fbf-2 gonads, all 1 day past mid-L4. Scale bars represent 50 μM. Gonads are outlined. Empty arrowhead marks distal end. (b) Nomarski micrograph of Fog germline with oocytes marked by solid arrowheads, shown in intact animal. The distal germline has many undifferentiated cells. Germlines of this appearance could be fertile and make viable progeny when crossed to wild-type males, showing that FBF is not required for oogenesis or embryogenesis. (c) Hoechst-stained germline from the “other sterile” group. The germline has distal undifferentiated germ cells and proximal germ cells that had deteriorated (outlined by solid line). (d,e) Distal region of same Fog gonad contains undifferentiated cells with nuclear morphology characteristic of mitotic cell cycle. (d) DAPI-stained. (e) EdU incorporation highlights actively cycling cells. (f,g) Same gonad with a tumorous germline. (f) DAPI-stained. (g) Stained with anti-phosphorylated histone H3 to mark nuclei of cells in late G2 and M-phase (41).
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Related In: Results  -  Collection

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Figure 3: Overexpressed FOG-3 blocks proliferation in fbf-1 fbf-2 mutants. (a) Germlines were scored for presence of undifferentiated cells in the distal gonad (Distal undiff) and for gamete formation in adults (Proximal germline); Mog (masculinization of germline), sperm only; Fog, oocytes only; Tum, undifferentiated throughout germline; other sterile. (b–g) Adult fog-3; fbf-1 fbf-2 gonads, all 1 day past mid-L4. Scale bars represent 50 μM. Gonads are outlined. Empty arrowhead marks distal end. (b) Nomarski micrograph of Fog germline with oocytes marked by solid arrowheads, shown in intact animal. The distal germline has many undifferentiated cells. Germlines of this appearance could be fertile and make viable progeny when crossed to wild-type males, showing that FBF is not required for oogenesis or embryogenesis. (c) Hoechst-stained germline from the “other sterile” group. The germline has distal undifferentiated germ cells and proximal germ cells that had deteriorated (outlined by solid line). (d,e) Distal region of same Fog gonad contains undifferentiated cells with nuclear morphology characteristic of mitotic cell cycle. (d) DAPI-stained. (e) EdU incorporation highlights actively cycling cells. (f,g) Same gonad with a tumorous germline. (f) DAPI-stained. (g) Stained with anti-phosphorylated histone H3 to mark nuclei of cells in late G2 and M-phase (41).
Mentions: To assess the effect of overexpressed FOG-3 on germline proliferation, we compared fbf-1 fbf-2 mutant germlines with and without FOG-3. For these studies, we focused on adults, 24 hours past the L4 stage, and first asked whether germ cells were undifferentiated or differentiated in the distal gonad (Figure 3a, Distal undiff). Prior work showed that the distal gonads of wild-type animals and fog-3 mutants maintain undifferentiated germ cells throughout adulthood (15, 25, 26), but that the distal gonads of late L4 fbf-1 fbf-2 larvae contain no undifferentiated germ cells because all their germ cells differentiate into sperm (21). We first confirmed these conclusions for the distal germlines of wild-type, fog-3 and fbf-1 fbf-2 late L4 larvae (Figure 3a, lines 1–3). We then examined the effect of removing FOG-3 in fbf-1 fbf-2 mutants. In fog-3; fbf-1 fbf-2 triple mutant adults, the distal germ cells all appeared undifferentiated (Figure 3a, line 4; Figures 3b and c). A similar effect was seen when FOG-3 was depleted using RNAi (Figure 3a, line 6) or reduced by one gene copy (Figure 3a, line 7). To ask if these distal undifferentiated cells in fog-3; fbf-1 fbf-2 germlines were cycling, we treated animals with the thymidine analog EdU, an S-phase marker, and found that nuclei among the undifferentiated germ cells were labeled (Figures 3d and e). Therefore, these undifferentiated nuclei are progressing through the cell cycle.

Bottom Line: Thus, fog-3 mutants possess fewer germ cells than normal, a modest but reproducible decrease observed for each of two distinct fog-3 alleles.Finally, we show that FOG-3 and FBF work together to promote tumor formation in animals carrying oncogenic Notch mutations.A similar effect was not observed when germline tumors were induced by manipulation of other regulators; therefore, this FOG-3 tumor-promoting effect is context dependent.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA.

ABSTRACT
Vertebrate Tob/BTG proteins inhibit cell proliferation when overexpressed in tissue-culture cells, and they can function as tumor suppressors in mice. The single Caenorhabditis elegans Tob/BTG ortholog, FOG-3, by contrast, was identified from its loss-of-function phenotype as a regulator of sperm fate specification. Here we report that FOG-3 also regulates proliferation in the germline tissue. We first demonstrate that FOG-3 is a positive regulator of germline proliferation. Thus, fog-3 mutants possess fewer germ cells than normal, a modest but reproducible decrease observed for each of two distinct fog-3 alleles. A similar decrease also occurred in fog-3/+ heterozygotes, again for both fog-3 alleles, revealing a haplo-insufficient effect on proliferation. Therefore, FOG-3 normally promotes proliferation, and two copies of the fog-3 gene are required for this function. We next overexpressed FOG-3 by removal of FBF, the collective term for FBF-1 and FBF-2, two nearly identical PUF RNA-binding proteins. We find that overexpressed FOG-3 blocks proliferation in fbf-1 fbf-2 mutants; whereas germ cells stop dividing and instead differentiate in fbf-1 fbf-2 double mutants, they continue to proliferate in fog-3; fbf-1 fbf-2 triple mutants. Therefore, like its vertebrate Tob/BTG cousins, overexpressed FOG-3 is 'antiproliferative'. Indeed, some fog-3; fbf-1 fbf-2 mutants possess small tumors, suggesting that FOG-3 can act as a tumor suppressor. Finally, we show that FOG-3 and FBF work together to promote tumor formation in animals carrying oncogenic Notch mutations. A similar effect was not observed when germline tumors were induced by manipulation of other regulators; therefore, this FOG-3 tumor-promoting effect is context dependent. We conclude that FOG-3 can either promote or inhibit proliferation in a manner that is sensitive to both genetic context and gene dosage. The discovery of these FOG-3 effects on proliferation has implications for our understanding of vertebrate Tob/BTG proteins and their influence on normal development and tumorigenesis.

Show MeSH
Related in: MedlinePlus