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STEM_790_sm_supplFig1.pdf257KSupplemental Figure 1. Strategy of Nanos2-overexpression by Ngn3-Cre. (A): A schematic drawing of the relationship between the typical morphology and gene expression patterns in PLZF-positive undifferentiated spermatogonia. Asingle and Apaired spermatogonia contain a higher proportion of GFRA1+NANOS2+, while many of Aalinged are NANOS3+Ngn3+, which then become differentiating spermatogonia. Of note, the heterogeneity exists even in the cysts with the same number of cells with regard to their gene expression (e.g. a small subset of Asingle spermatogonia are GFRA1-NANOS2-NANOS3+Ngn3+). (B): Rosa-YFP reporter mice start to express YFP after removal of the inactivating sequence by Cre-mediated recombination. Because the recombination is irreversible and inheritable, progenies of the recombined cells can be marked by YFP. (C): A conditional transgenic mouse line, CAGfloxed CAT-3xFlag-Nanos2 (Tg-Nanos2) is capable of expressing Flag tagged-Nanos2 under the control of the ubiquitous CAG promoter after excision of the floxed CAT gene (an innocent gene) by Cre recombinase. (D): The experimental schedule. Ngn3-Cre starts to be expressed in spermatogonia around P7. We collected testes at the indicated time points. Abbreviations: PLZF, promyelocytic leukemia zinc-finger; GFRA1, glial cell line-derived neurotrophic factor family receptor alpha 1; Ngn3, Neurogenin3; Tg, transgene; CAG, cytomegalovirus (CMV) early enhancer/chicken beta-actin promoter; CAT, chloramphenicol acetyltransferase; P, postnatal day.
STEM_790_sm_supplFig2.pdf260KSupplemental Figure 2. Ngn3-Cre targets GFRA1-negative cells. (A-F): At P7, Rosa-YFP; Ngn3-Cre double transgenic testes were immunostained with the indicated markers. Most of YFP-positive spermatogonia (Ngn3-lineage cells) were not stained with GFRA1. Scale bar, 100 μm. (G): Quantification of YFP-positive cells per GFRA1-positive cells at P7 and 6 weeks (N=3). Abbreviations: GFRA1, glial cell line-derived neurotrophic factor family receptor alpha 1; Ngn3, Neurogenin3; P, postnatal day.
STEM_790_sm_supplFig3.pdf311KSupplemental Figure 3. Nanos2 overexpressed by Ngn3-Cre did not induce de novo GFRA1 expression. (A-I): Immunostaining of 6-week-old Tg-Nanos2; Ngn3-Cre double transgenic testes with the indicated markers. Most of FLAG-positive cells did not express GFRA1. Scale bar, 100 μm. (J-K): Quantification of GFRA1-positive cells per FLAG- (J) or PLZF- (K) positive cells in 6-week-old mice (N=3). The proportion of GFRA1-positive cells was not changed after Ngn3-Cre driven Nanos2-overexpression. Abbreviations: GFRA1, glial cell line-derived neurotrophic factor family receptor alpha 1; PLZF, promyelocytic leukemia zincfinger; Tg, transgene; Ngn3, Neurogenin3.
STEM_790_sm_supplFig4.pdf175KSupplemental Figure 4. Strategy of Nanos2-overexpression in the background of Gfra1- conditional knockout. (A): Floxed Gfra1 allele expresses Gfra1, thereby serving as a functional allele. Activation of Cre recombinase results in a removal of floxed Gfra1 sequences, simultaneously generating EGFP knock-in (Gfra1 knock-out) allele. (B): A conditional transgenic mouse line, CAG-floxed CAT-3xFlag-Nanos2 (Tg-Nanos2) is capable of expressing Flag tagged-Nanos2 under the control of the ubiquitous CAG promoter after excision of the floxed CAT gene (an innocent gene) by Cre recombinase. (C): The experimental schedule. Cre activity was induced by intraperitoneal injection of Tamoxifen into 4-week-old mice for 5 consecutive days and the testes were harvested at the indicated time points. Abbreviations: GFRA1, glial cell line-derived neurotrophic factor family receptor alpha 1; Tg, transgene; CAG, cytomegalovirus (CMV) early enhancer/chicken beta-actin promoter; CAT, chloramphenicol acetyltransferase.
STEM_790_sm_supplFig5.pdf394KSupplemental Figure 5. Successful induction of Nanos2-overexpression by Cre-Ert2. (A-I): Five-week-old testes were immunostained with the indicated markers. In control Gfra1δ/+ mice, NANOS2 was expressed in a small subset of spermatogonia (arrowheads). In Gfra1δ/+; Tg-Nanos2 or Gfra1δ/δ; Tg-Nanos2 mice, strong NANOS2 signal were detected in early germ cells located on the basal side of seminiferous tubules. Scale bar, 100 μm. Abbreviations: GFRA1, glial cell line-derived neurotrophic factor family receptor alpha 1; Tg, transgene.
STEM_790_sm_supplFig6.pdf341KSupplemental Figure 6. Partial rescue of Gfra1-conditional knockout phenotype by overexpressed Nanos2. (A-H): Eight-week-old testes were stained with indicated markers. PLZF- or GFP- positive spermatogonia are indicated by arrowheads. At 8-weeks, both PLZF- and GFP- positive spermatogonia were observed in Gfra1δ/δ; Tg-Nanos2 mice but not in Gfra1δ/δ mice. Scale bar, 100 μm. Abbreviations: PLZF, promyelocytic leukemia zinc-finger; GFRA1, glial cell line-derived neurotrophic factor family receptor alpha 1; Tg, transgene.
STEM_790_sm_supplFig7.pdf273KSupplemental Figure 7. Apoptotic cell death in each mutant. (A-D): The representative images of double immunostaining with anti-GFP (green, A-D) or anti-SCP3 (a marker of meiosis, green, E-G) and anti-cleaved PARP (magenta, A-G) antibodies at 5 weeks. Arrowheads indicate GFP-positive cells. Apoptotic cells were increased in Nanos2- overexpressing testes especially in meiotic germ cells (E-G), but the most of them were GFPnegative (A-D). Scale bar, 100 μm. Abbreviations: GFRA1, glial cell line-derived neurotrophic factor family receptor alpha 1; Tg, transgene; SCP3, synaptonemal complex protein 3; PARP, poly (ADP-ribose) polymerase.
STEM_790_sm_supplFig8.pdf290KSupplemental Figure 8. Detection of cell proliferation in Gfra1-conditional knockout and Nanos2-overexpressing cells. (A-H): The representative images of double immunostaining with anti-GFP (green) and anti-PH3 (magenta) antibodies at 5 weeks. GFP-positive spermatogonia are indicated by arrowheads. Scale bar, 100 μm. Abbreviations: GFRA1, glial cell line-derived neurotrophic factor family receptor alpha 1; Tg, transgene; PH3, phospho Histone H3.
STEM_790_sm_supplFig9.pdf157KSupplemental Figure 9. Quantification of proliferation and apoptotic cell death in PLZFpositive spermatogonia. Quantification of apoptotic (A) and proliferative (B) spermatogonia in 5-week-old mice (N≧3). **, P<0.01 and *, P<0.05. Apoptosis in the PLZF-positive cells didn't increase in any mutants (A), indicating that the mutant cells survived even after losing GFP expression. However, we did not find much difference in proliferation rate between control and Gfra1δ/δ testes in PLZF-positive population (B). Since the ratio of GFP-positive cells in PLZF-positive cells is low (20-25%), the decrease in the proliferation rate in the GFP-positive cells in Gfra1δ/δ was not reflected in the proliferation rate in the PLZF-positive population. Abbreviations: PLZF, promyelocytic leukemia zinc-finger; PARP, poly (ADPribose) polymerase; PH3, phospho Histone H3; GFRA1, glial cell line-derived neurotrophic factor family receptor alpha 1; Tg, transgene.
STEM_790_sm_supplFig10.pdf168KSupplemental Figure 10. Quantification of proliferation and apoptotic cell death at 8-weeks of age. Quantification of apoptotic (A) and proliferative (B) spermatogonia in 8-week-old mice (N4). **, P<0.01. No GFP-positive spermatogonia are observed in Gfra1δ/δ mice at 8- weeks. Abbreviations: GFRA1, glial cell line-derived neurotrophic factor family receptor alpha 1; Tg, transgene; PARP, poly (ADP-ribose) polymerase; PH3, phospho Histone H3.
STEM_790_sm_supplFig11.pdf216KSupplemental Figure 11. Overexpression of Nanos2 by Cre-Ert2 suppresses differentiation of PLZF-positive spermatogonia. (A-D): Whole-mount immunostaining of 5-week-old testes with the indicated markers. Scale bar, 100 μm. (E): Frequency of NANOS3-positive spermatogonial clusters (2n cells: 1, 2, 4, 8, 16) were defined in PLZF-positive population (N=3). **, P<0.01. Differentiation rate in PLZF-positive cells in Gfra1δ/δ testes were increased as expected by the promoted differentiation in GFP-positive cells, while it was strongly suppressed by Nanos2-overexpression. Abbreviations: PLZF, promyelocytic leukemia zinc-finger; GFRA1, glial cell line-derived neurotrophic factor family receptor alpha 1; As, Asingle ; Apr, Apaired; Aal, Aaligned; Tg, transgene.
STEM_790_sm_supplFig12.pdf603KSupplemental Figure 12. Summary of results. In the wild-type, undifferentiated spermatogonia can be classified at least two groups according to the cellular state and the marker gene expression: one is NANOS2+GFRA1+ (yellow) and the other is NANOS3+Ngn3+ (blue). (A): In normal NANOS2+GFRA1+ cells (yellow), NANOS2 suppresses differentiation in the downstream of GFRA1. The proliferation of these cells is balanced by a positive regulation of GDNF signaling and a negative regulation of NANOS2. After losing NANOS2/GFRA1 expressions (blue), spermatogonia might be in more mature state, and ready for the next steps of differentiation (red right arrow). (B): NANOS2 induced by Nanos3-Cre blocks differentiation of NANOS2+GFRA1+ cells (yellow) into NANOS3+Ngn3+ state (blue). (C): NANOS2 is induced only in NANOS3+Ngn3+ cells (blue) by Ngn3-Cre. Both differentiation and proliferation are suppressed even in the absence of GFRA1. NANOS2+GFRA1+ population (yellow) is normally maintained and enters into NANOS3+Ngn3+ state. (D): In Gfra1-cKO mice, differentiation-related genes, such as NANOS3 is abnormally up-regulated in NANOS2+GFRA1+ spermatogonia (yellow), which may cause precocious activation of differentiation pathway (red right arrow) and loss of stem cell population. (E): Nanos2-overexpression by Cre-Ert2 suppresses differentiation and proliferation of both NANOS2+GFRA1+ (yellow) and NANOS3+Ngn3+ (blue) population. (F): Nanos2-overexpression can block differentiation progression of Gfra1-null cells (yellow) and delay the stem cell loss phenotypes of these mice. However, spermatogonial population is eventually lost because of a strikingly lower level of proliferation and a constant level of apoptotic cell death. Proliferation of NANOS3+Ngn3+ cells is supported by unknown growth factor (X) in the absence of GFRA1. Abbreviations: GFRA1, glial cell line-derived neurotrophic factor family receptor alpha 1; OE, overexpression; Tg, transgene; Ngn3, Neurogenin3.

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