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Hierarchical differentiation competence in response to retinoic acid ensures stem cell maintenance during mouse spermatogenesis.

Ikami K, Tokue M, Sugimoto R, Noda C, Kobayashi S, Hara K, Yoshida S - Development (2015)

Bottom Line: Ectopic expression of RARγ was sufficient to induce GFRα1(+) cells to directly differentiate to KIT(+) cells without transiting the NGN3(+) state.Therefore, RARγ plays key roles in the differentiation competence of NGN3(+) cells.We propose a novel mechanism of stem cell fate selection in an open niche environment whereby undifferentiated cells show heterogeneous competence to differentiate in response to ubiquitously distributed differentiation-inducing signals.

View Article: PubMed Central - PubMed

Affiliation: Division of Germ Cell Biology, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies (Sokendai), 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.

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Fate of pulse-labeled GFRα1+ spermatogonia during VAD/VA administration. (A) The experimental schedule for B and C. Two days after the TM pulse, Gfra1-CreERT2; CAG-CAT-EGFP transgenic mice maintained in VAD were injected with VA and then fed a normal diet. Testis samples were harvested at the indicated times. (B) Representative IF images of whole-mount seminiferous tubule 0, 2, 6 and 10 days after VA injection stained for GFP and GFRα1. (C) The number of GFP-labeled GFRα1+ Aundiff (magenta), GFRα1− Aundiff (green), KIT+ (blue) spermatogonia and total labeled (black) cells. Shown is the mean±s.e.m. of five, five, seven, six, four and three testes for days 0, 2, 4, 6, 8 and 10, respectively. Data, except for those of GFRα1+ cells, on days 2, 6, 8 and 10 were significantly different compared with the values on day 0 (P<0.03, t-test). (D) Schedule for E and F. After the TM pulse, Gfra1-CreERT2; CAG-CAT-EGFP transgenic mice were continually fed the VAD diet. (E) Representative IF images of whole-mount seminiferous tubule 2, 14 and 30 days after TM pulse stained for GFP and GFRα1. (F) Number of GFP-labeled GFRα1+ Aundiff (magenta) and GFRα1− Aundiff (green) spermatogonia and total labeled cells (black), shown as the mean±s.e.m. of eight, three and five testes on days 0, 14 and 30, respectively. *P=0.032, **P<0.002 (t-test) compared with the values for day 2. Scale bars: 50 μm.
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DEV118695F3: Fate of pulse-labeled GFRα1+ spermatogonia during VAD/VA administration. (A) The experimental schedule for B and C. Two days after the TM pulse, Gfra1-CreERT2; CAG-CAT-EGFP transgenic mice maintained in VAD were injected with VA and then fed a normal diet. Testis samples were harvested at the indicated times. (B) Representative IF images of whole-mount seminiferous tubule 0, 2, 6 and 10 days after VA injection stained for GFP and GFRα1. (C) The number of GFP-labeled GFRα1+ Aundiff (magenta), GFRα1− Aundiff (green), KIT+ (blue) spermatogonia and total labeled (black) cells. Shown is the mean±s.e.m. of five, five, seven, six, four and three testes for days 0, 2, 4, 6, 8 and 10, respectively. Data, except for those of GFRα1+ cells, on days 2, 6, 8 and 10 were significantly different compared with the values on day 0 (P<0.03, t-test). (D) Schedule for E and F. After the TM pulse, Gfra1-CreERT2; CAG-CAT-EGFP transgenic mice were continually fed the VAD diet. (E) Representative IF images of whole-mount seminiferous tubule 2, 14 and 30 days after TM pulse stained for GFP and GFRα1. (F) Number of GFP-labeled GFRα1+ Aundiff (magenta) and GFRα1− Aundiff (green) spermatogonia and total labeled cells (black), shown as the mean±s.e.m. of eight, three and five testes on days 0, 14 and 30, respectively. *P=0.032, **P<0.002 (t-test) compared with the values for day 2. Scale bars: 50 μm.

Mentions: Next, we determined the fate of GFRα1+ spermatogonia in the same VAD model, using Gfra1-CreERT2; CAG-CAT-EGFP mice (Fig. 3A). Two days after pulse under VAD, the majority of the labeled cells were GFRα1+, as expected. The response to VA differed to that of NGN3+ cells in that the cells proliferated and the total number of labeled cells, which included a constant number of GFRα1+ cells, increased (Fig. 3B,C; supplementary material Fig. S2A,B). Simultaneously, the number of labeled cells that were KIT−/GFRα1− (which largely represented NGN3+ cells) increased slowly until day 8, indicating that GFRα1+ cells generated NGN3+ cells while maintaining the size of the GFRα1+ population. Between days 8 and 10 after VA administration, the number of these labeled KIT−/GFRα1− cells decreased significantly, whereas the number of labeled KIT+ cells increased (Fig. 3C). This reflects the second round of NGN3+ to KIT+ differentiation, which occurs when the seminiferous epithelium returns to stages VIII to IX after one cycle of 8.6 days (Sugimoto et al., 2012).Fig. 3.


Hierarchical differentiation competence in response to retinoic acid ensures stem cell maintenance during mouse spermatogenesis.

Ikami K, Tokue M, Sugimoto R, Noda C, Kobayashi S, Hara K, Yoshida S - Development (2015)

Fate of pulse-labeled GFRα1+ spermatogonia during VAD/VA administration. (A) The experimental schedule for B and C. Two days after the TM pulse, Gfra1-CreERT2; CAG-CAT-EGFP transgenic mice maintained in VAD were injected with VA and then fed a normal diet. Testis samples were harvested at the indicated times. (B) Representative IF images of whole-mount seminiferous tubule 0, 2, 6 and 10 days after VA injection stained for GFP and GFRα1. (C) The number of GFP-labeled GFRα1+ Aundiff (magenta), GFRα1− Aundiff (green), KIT+ (blue) spermatogonia and total labeled (black) cells. Shown is the mean±s.e.m. of five, five, seven, six, four and three testes for days 0, 2, 4, 6, 8 and 10, respectively. Data, except for those of GFRα1+ cells, on days 2, 6, 8 and 10 were significantly different compared with the values on day 0 (P<0.03, t-test). (D) Schedule for E and F. After the TM pulse, Gfra1-CreERT2; CAG-CAT-EGFP transgenic mice were continually fed the VAD diet. (E) Representative IF images of whole-mount seminiferous tubule 2, 14 and 30 days after TM pulse stained for GFP and GFRα1. (F) Number of GFP-labeled GFRα1+ Aundiff (magenta) and GFRα1− Aundiff (green) spermatogonia and total labeled cells (black), shown as the mean±s.e.m. of eight, three and five testes on days 0, 14 and 30, respectively. *P=0.032, **P<0.002 (t-test) compared with the values for day 2. Scale bars: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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DEV118695F3: Fate of pulse-labeled GFRα1+ spermatogonia during VAD/VA administration. (A) The experimental schedule for B and C. Two days after the TM pulse, Gfra1-CreERT2; CAG-CAT-EGFP transgenic mice maintained in VAD were injected with VA and then fed a normal diet. Testis samples were harvested at the indicated times. (B) Representative IF images of whole-mount seminiferous tubule 0, 2, 6 and 10 days after VA injection stained for GFP and GFRα1. (C) The number of GFP-labeled GFRα1+ Aundiff (magenta), GFRα1− Aundiff (green), KIT+ (blue) spermatogonia and total labeled (black) cells. Shown is the mean±s.e.m. of five, five, seven, six, four and three testes for days 0, 2, 4, 6, 8 and 10, respectively. Data, except for those of GFRα1+ cells, on days 2, 6, 8 and 10 were significantly different compared with the values on day 0 (P<0.03, t-test). (D) Schedule for E and F. After the TM pulse, Gfra1-CreERT2; CAG-CAT-EGFP transgenic mice were continually fed the VAD diet. (E) Representative IF images of whole-mount seminiferous tubule 2, 14 and 30 days after TM pulse stained for GFP and GFRα1. (F) Number of GFP-labeled GFRα1+ Aundiff (magenta) and GFRα1− Aundiff (green) spermatogonia and total labeled cells (black), shown as the mean±s.e.m. of eight, three and five testes on days 0, 14 and 30, respectively. *P=0.032, **P<0.002 (t-test) compared with the values for day 2. Scale bars: 50 μm.
Mentions: Next, we determined the fate of GFRα1+ spermatogonia in the same VAD model, using Gfra1-CreERT2; CAG-CAT-EGFP mice (Fig. 3A). Two days after pulse under VAD, the majority of the labeled cells were GFRα1+, as expected. The response to VA differed to that of NGN3+ cells in that the cells proliferated and the total number of labeled cells, which included a constant number of GFRα1+ cells, increased (Fig. 3B,C; supplementary material Fig. S2A,B). Simultaneously, the number of labeled cells that were KIT−/GFRα1− (which largely represented NGN3+ cells) increased slowly until day 8, indicating that GFRα1+ cells generated NGN3+ cells while maintaining the size of the GFRα1+ population. Between days 8 and 10 after VA administration, the number of these labeled KIT−/GFRα1− cells decreased significantly, whereas the number of labeled KIT+ cells increased (Fig. 3C). This reflects the second round of NGN3+ to KIT+ differentiation, which occurs when the seminiferous epithelium returns to stages VIII to IX after one cycle of 8.6 days (Sugimoto et al., 2012).Fig. 3.

Bottom Line: Ectopic expression of RARγ was sufficient to induce GFRα1(+) cells to directly differentiate to KIT(+) cells without transiting the NGN3(+) state.Therefore, RARγ plays key roles in the differentiation competence of NGN3(+) cells.We propose a novel mechanism of stem cell fate selection in an open niche environment whereby undifferentiated cells show heterogeneous competence to differentiate in response to ubiquitously distributed differentiation-inducing signals.

View Article: PubMed Central - PubMed

Affiliation: Division of Germ Cell Biology, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies (Sokendai), 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.

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Related in: MedlinePlus