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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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Ectopic RARγ expression by GFRα1+ spermatogonia. (A) The CAG-CAT-3xFLAG-Rarg transgene. When CAT between the loxP sites is deleted by TM-activated Cre, FLAG-tagged RARγ is constitutively expressed under the control of the CAG promoter. (B) Experimental design of the fate analysis of GFRα1+ cells with enforced FLAG-RARγ expression upon VA readministration in VAD mice, as shown in C-F. Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg transgenic mice were maintained in VAD and VA was administered 2 days after TM injection, as indicated. Testes were then processed for IF. (C,D) IF images of whole-mount seminiferous tubules of the mice described above, 2 days after VA injection, stained for FLAG-RARγ (green) and KIT (magenta) (C), and cell number relative to the number of initial induced cells (D). Data for GFP-labeled NGN3+ and GFRα1+ cells are reproduced from Fig. 2C and Fig. 3C, respectively, for comparison. The mean±s.e.m. value of three testes is shown. *P<0.003 (t-test), compared with the values of FLAG-RARγ+ GFRα1+ cells at day 2. (E,F) Representative confocal images of the same field of whole-mounts of seminiferous tubules of mice treated as described above, at 2 days after VA injection; staining was performed for GFRα1, KIT and FLAG (E). Open arrowheads, white arrowheads and small arrows indicate FLAG+ cells that are GFRα1+/KIT+, GFRα1+/KIT− and GFRα1−/KIT+, respectively. (F) Quantitation of GFP+ and FLAG-RARγ+ cells showing different patterns of GFRα1 and KIT expression in Gfra1-CreERT2; CAG-CAT-EGFP and Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg mice, respectively. Cell numbers are shown above each bar. (G) Experimental design of the fate analysis of GFRα1+ cells with enforced FLAG-RARγ expression under normal conditions, as shown in H-J. Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg transgenic mice were pulsed with TM at 13-17 weeks of age, and after 2 and 10 days their testes were processed for IF. (H) IF images of whole-mount seminiferous tubules 2 and 10 days after TM injection, stained for FLAG-RARγ and GFRα1. (I,J) Numbers of GFRα1+ Aundiff (magenta), GFRα1− Aundiff (green), KIT+ (blue) spermatogonia and total cells (black) in either GFP-labeled (I) or FLAG-RARγ-expressing (J) cells of Gfra1-CreERT2; CAG-CAT-EGFP and Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg mice, respectively, following the schedule shown in G. The mean±s.e.m. of four (I) and three (J) testes are shown. *P<0.05 (t-test), compared with the values on day 2. Scale bars: 50 μm.
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DEV118695F5: Ectopic RARγ expression by GFRα1+ spermatogonia. (A) The CAG-CAT-3xFLAG-Rarg transgene. When CAT between the loxP sites is deleted by TM-activated Cre, FLAG-tagged RARγ is constitutively expressed under the control of the CAG promoter. (B) Experimental design of the fate analysis of GFRα1+ cells with enforced FLAG-RARγ expression upon VA readministration in VAD mice, as shown in C-F. Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg transgenic mice were maintained in VAD and VA was administered 2 days after TM injection, as indicated. Testes were then processed for IF. (C,D) IF images of whole-mount seminiferous tubules of the mice described above, 2 days after VA injection, stained for FLAG-RARγ (green) and KIT (magenta) (C), and cell number relative to the number of initial induced cells (D). Data for GFP-labeled NGN3+ and GFRα1+ cells are reproduced from Fig. 2C and Fig. 3C, respectively, for comparison. The mean±s.e.m. value of three testes is shown. *P<0.003 (t-test), compared with the values of FLAG-RARγ+ GFRα1+ cells at day 2. (E,F) Representative confocal images of the same field of whole-mounts of seminiferous tubules of mice treated as described above, at 2 days after VA injection; staining was performed for GFRα1, KIT and FLAG (E). Open arrowheads, white arrowheads and small arrows indicate FLAG+ cells that are GFRα1+/KIT+, GFRα1+/KIT− and GFRα1−/KIT+, respectively. (F) Quantitation of GFP+ and FLAG-RARγ+ cells showing different patterns of GFRα1 and KIT expression in Gfra1-CreERT2; CAG-CAT-EGFP and Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg mice, respectively. Cell numbers are shown above each bar. (G) Experimental design of the fate analysis of GFRα1+ cells with enforced FLAG-RARγ expression under normal conditions, as shown in H-J. Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg transgenic mice were pulsed with TM at 13-17 weeks of age, and after 2 and 10 days their testes were processed for IF. (H) IF images of whole-mount seminiferous tubules 2 and 10 days after TM injection, stained for FLAG-RARγ and GFRα1. (I,J) Numbers of GFRα1+ Aundiff (magenta), GFRα1− Aundiff (green), KIT+ (blue) spermatogonia and total cells (black) in either GFP-labeled (I) or FLAG-RARγ-expressing (J) cells of Gfra1-CreERT2; CAG-CAT-EGFP and Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg mice, respectively, following the schedule shown in G. The mean±s.e.m. of four (I) and three (J) testes are shown. *P<0.05 (t-test), compared with the values on day 2. Scale bars: 50 μm.

Mentions: We performed a gain-of-function experiment by ectopic expression of RARγ in GFRα1+ spermatogonia. We generated Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg mice, in which FLAG-tagged RARγ can be induced in GFRα1+ cells upon TM injection (Fig. 5A; supplementary material Fig. S4A,B). The behavior of GFRα1+ cells that expressed FLAG-RARγ was first tested in the VAD/VA administration model (Fig. 5B-F). In VAD (day 0), most of the FLAG-RARγ+ cells were GFRα1+ and KIT−, similar to the control GFP-labeled GFRα1+ cells (Fig. 3). However, when VA was replaced, FLAG-RARγ+ cells became KIT+ in 2 days, in stark contrast to GFP-labeled GFRα1+ cells, which largely remained KIT− (Fig. 5C,D and Fig. 3C). These results clearly indicate that enforced RARγ expression provides GFRα1+ spermatogonia with the competence to differentiate into KIT+ spermatogonia, which is embodied when the RA signal is generated.Fig. 5.


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)

Ectopic RARγ expression by GFRα1+ spermatogonia. (A) The CAG-CAT-3xFLAG-Rarg transgene. When CAT between the loxP sites is deleted by TM-activated Cre, FLAG-tagged RARγ is constitutively expressed under the control of the CAG promoter. (B) Experimental design of the fate analysis of GFRα1+ cells with enforced FLAG-RARγ expression upon VA readministration in VAD mice, as shown in C-F. Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg transgenic mice were maintained in VAD and VA was administered 2 days after TM injection, as indicated. Testes were then processed for IF. (C,D) IF images of whole-mount seminiferous tubules of the mice described above, 2 days after VA injection, stained for FLAG-RARγ (green) and KIT (magenta) (C), and cell number relative to the number of initial induced cells (D). Data for GFP-labeled NGN3+ and GFRα1+ cells are reproduced from Fig. 2C and Fig. 3C, respectively, for comparison. The mean±s.e.m. value of three testes is shown. *P<0.003 (t-test), compared with the values of FLAG-RARγ+ GFRα1+ cells at day 2. (E,F) Representative confocal images of the same field of whole-mounts of seminiferous tubules of mice treated as described above, at 2 days after VA injection; staining was performed for GFRα1, KIT and FLAG (E). Open arrowheads, white arrowheads and small arrows indicate FLAG+ cells that are GFRα1+/KIT+, GFRα1+/KIT− and GFRα1−/KIT+, respectively. (F) Quantitation of GFP+ and FLAG-RARγ+ cells showing different patterns of GFRα1 and KIT expression in Gfra1-CreERT2; CAG-CAT-EGFP and Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg mice, respectively. Cell numbers are shown above each bar. (G) Experimental design of the fate analysis of GFRα1+ cells with enforced FLAG-RARγ expression under normal conditions, as shown in H-J. Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg transgenic mice were pulsed with TM at 13-17 weeks of age, and after 2 and 10 days their testes were processed for IF. (H) IF images of whole-mount seminiferous tubules 2 and 10 days after TM injection, stained for FLAG-RARγ and GFRα1. (I,J) Numbers of GFRα1+ Aundiff (magenta), GFRα1− Aundiff (green), KIT+ (blue) spermatogonia and total cells (black) in either GFP-labeled (I) or FLAG-RARγ-expressing (J) cells of Gfra1-CreERT2; CAG-CAT-EGFP and Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg mice, respectively, following the schedule shown in G. The mean±s.e.m. of four (I) and three (J) testes are shown. *P<0.05 (t-test), compared with the values on day 2. Scale bars: 50 μm.
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DEV118695F5: Ectopic RARγ expression by GFRα1+ spermatogonia. (A) The CAG-CAT-3xFLAG-Rarg transgene. When CAT between the loxP sites is deleted by TM-activated Cre, FLAG-tagged RARγ is constitutively expressed under the control of the CAG promoter. (B) Experimental design of the fate analysis of GFRα1+ cells with enforced FLAG-RARγ expression upon VA readministration in VAD mice, as shown in C-F. Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg transgenic mice were maintained in VAD and VA was administered 2 days after TM injection, as indicated. Testes were then processed for IF. (C,D) IF images of whole-mount seminiferous tubules of the mice described above, 2 days after VA injection, stained for FLAG-RARγ (green) and KIT (magenta) (C), and cell number relative to the number of initial induced cells (D). Data for GFP-labeled NGN3+ and GFRα1+ cells are reproduced from Fig. 2C and Fig. 3C, respectively, for comparison. The mean±s.e.m. value of three testes is shown. *P<0.003 (t-test), compared with the values of FLAG-RARγ+ GFRα1+ cells at day 2. (E,F) Representative confocal images of the same field of whole-mounts of seminiferous tubules of mice treated as described above, at 2 days after VA injection; staining was performed for GFRα1, KIT and FLAG (E). Open arrowheads, white arrowheads and small arrows indicate FLAG+ cells that are GFRα1+/KIT+, GFRα1+/KIT− and GFRα1−/KIT+, respectively. (F) Quantitation of GFP+ and FLAG-RARγ+ cells showing different patterns of GFRα1 and KIT expression in Gfra1-CreERT2; CAG-CAT-EGFP and Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg mice, respectively. Cell numbers are shown above each bar. (G) Experimental design of the fate analysis of GFRα1+ cells with enforced FLAG-RARγ expression under normal conditions, as shown in H-J. Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg transgenic mice were pulsed with TM at 13-17 weeks of age, and after 2 and 10 days their testes were processed for IF. (H) IF images of whole-mount seminiferous tubules 2 and 10 days after TM injection, stained for FLAG-RARγ and GFRα1. (I,J) Numbers of GFRα1+ Aundiff (magenta), GFRα1− Aundiff (green), KIT+ (blue) spermatogonia and total cells (black) in either GFP-labeled (I) or FLAG-RARγ-expressing (J) cells of Gfra1-CreERT2; CAG-CAT-EGFP and Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg mice, respectively, following the schedule shown in G. The mean±s.e.m. of four (I) and three (J) testes are shown. *P<0.05 (t-test), compared with the values on day 2. Scale bars: 50 μm.
Mentions: We performed a gain-of-function experiment by ectopic expression of RARγ in GFRα1+ spermatogonia. We generated Gfra1-CreERT2; CAG-CAT-3xFLAG-Rarg mice, in which FLAG-tagged RARγ can be induced in GFRα1+ cells upon TM injection (Fig. 5A; supplementary material Fig. S4A,B). The behavior of GFRα1+ cells that expressed FLAG-RARγ was first tested in the VAD/VA administration model (Fig. 5B-F). In VAD (day 0), most of the FLAG-RARγ+ cells were GFRα1+ and KIT−, similar to the control GFP-labeled GFRα1+ cells (Fig. 3). However, when VA was replaced, FLAG-RARγ+ cells became KIT+ in 2 days, in stark contrast to GFP-labeled GFRα1+ cells, which largely remained KIT− (Fig. 5C,D and Fig. 3C). These results clearly indicate that enforced RARγ expression provides GFRα1+ spermatogonia with the competence to differentiate into KIT+ spermatogonia, which is embodied when the RA signal is generated.Fig. 5.

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