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First published online 23 January 2008
doi: 10.1242/dev.013474

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AKT signaling promotes derivation of embryonic germ cells from primordial germ cells

¹ Department of Pathology, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
² Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
³ Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan.

View larger version (46K): [in this window] [in a new window]   Fig. 1. PGCs of AKT-MER transgenic mice. (A) AKT-MER transgenic mice (right) and littermate control mice (left) at E11.5. As the Akt-Mer cDNA is linked to IRES-EGFP, the transgenic mice can be identified by EGFP fluorescence. (B) Flow cytometry analysis of cells from the gonads of E11.5 AKT-MER mice. Suspensions were stained for the PGC-specific marker SSEA-1. EGFP fluorescence was detected in the majority of the transgenic PGCs. (C) Level of AKT activation in the cultured PGCs. The E11.5 PGCs were seeded onto SCF-expressing Sl/Sl4-m220 feeder cells and cultured with or without bFGF and 4OHT for 1 day. The cells were stained with the SSEA-1 (green) and anti-Ser437-phosphorylated-AKT (pAKT) (red) antibodies. The fluorescence intensity of phospho-AKT in the individual PGCs was measured using LSM5 PASCAL confocal microscopy. Relative fluorescence per cell is shown in the right panel (mean±s.e.m.). The background fluorescence level was measured in the samples that were not treated with pAKT antibody. Phospho-AKT signals were stronger in the 4OHT-treated transgenic PGCs than in the untreated transgenic and the bFGF-treated wild-type controls (*P<0.01, #P<0.05, Student's t-test). The signals were stronger in the transgenic PGCs than in the control PGCs, even in the absence of 4OHT and bFGF (#P<0.05).

View larger version (38K): [in this window] [in a new window]   Fig. 2. EG cell production from AKT-MER-expressing PGCs under standard conditions. (A) Procedure for EG cell derivation. Cell suspensions of E11.5 gonads were seeded onto Sl/Sl4-m220 feeder cells that express the membrane-bound form of SCF, and cultured with LIF and bFGF (primary culture). 4OHT and bFGF were added only during primary culture. At day 5 of primary culture, whole wells were trypsinized and passaged onto fresh Sl/Sl4-m220 layers. Secondary culture was carried out with LIF for 5 days. The whole wells were finally passaged onto MEFs and cultured with LIF (tertiary culture). At day 5 of tertiary culture, the wells containing EG cells were counted to determine the derivation efficiency. The EG cells at tertiary culture were propagated to stable cell lines on MEFs in the presence of LIF. A fraction of EG cells in the primary cultures (primary EG cells) gave rise to stable EG cell lines by this whole-well passage procedure. The efficiency is detailed in the legend of Table 2A. (B) Alkaline phosphatase staining of primary EG cell colonies derived from wild-type PGCs cultured without 4OHT (left) and of transgenic PGCs cultured with 100 nM 4OHT (right). The EG cell colonies were multilayered and positive for alkaline phosphatase activity. Scale bar, 100 µm. (C) Increased number of AKT-MER-expressing PGCs. The cells of 0.02 embryos were added to each well, and 30-40 PGCs attached to feeder cells. The percentage change in the PGC population was calculated by dividing the number of PGCs at day 3 by the number of attached PGCs. The 4OHT-treated transgenic PGCs proliferated significantly more than untreated transgenic PGCs (mean±s.e.m.; *P<0.005, #P<0.0005, Student's t-test; n=6) and wild-type PGCs cultured with the corresponding concentrations of 4OHT (P<0.005, P<0.0005 and P<0.005 for 100 nM, 300 nM and 1000 nM, respectively). (D) Increased primary EG cell colony formation by E11.5 AKT-MER-expressing PGCs. A primary EG cell colony was defined as a multilayered, alkaline phosphatase-positive colony of more than 20 cells, as described previously (Kimura et al., 2003; Moe-Behrens et al., 2003). Colony formation was significantly higher for 4OHT-treated transgenic PGCs than for untreated transgenic PGCs (mean±s.e.m.; *P<0.005, #P<0.0005, Student's t-test; n=6) and wild-type PGCs cultured with the corresponding concentrations of 4OHT (P<0.0005, P<0.005 and P<0.0005 for 100 nM, 300 nM and 1000 nM, respectively).

View larger version (55K): [in this window] [in a new window]   Fig. 3. EG cell production from AKT-MER-expressing PGCs in the absence of bFGF and SCF. (A) Increased number of AKT-MER-expressing PGCs in the absence of bFGF. E11.5 gonad cell suspensions were seeded onto Sl/Sl4-m220 feeder cells and cultured with LIF but without bFGF. The cells of 0.1 embryos were added to each well. The percentage change in PGC number was calculated as described in Fig. 2C. The 4OHT-treated transgenic PGCs proliferated significantly more than did the untreated transgenic PGCs (mean±s.e.m.; *P<0.005, Student's t-test; n=4) and wild-type PGCs cultured with the corresponding concentrations of 4OHT (P<0.005 for 100 nM, 300 nM and 1000 nM). (B) Primary EG cell colony formation by E11.5 PGCs of transgenic mice without bFGF. Significantly more EG colonies were generated in the 4OHT-treated transgenic cultures than in the untreated transgenic PGCs (mean±s.e.m.; #P<0.001, *P<0.005, $P<0.05, Student's t-test; n=4). (C) Alkaline phosphatase-positive primary EG cell colonies generated from transgenic PGCs treated with 300 nM 4OHT in the absence of bFGF. Cell suspensions of transgenic gonads at E11.5 were seeded onto Sl/Sl4-m220 feeder cells and cultured with LIF alone for 5 days. Scale bar: 100 µm. (D) Transgenic EG cell lines established without bFGF from the primary PGC cultures in C. The EG cells formed round, multilayered colonies (left) and were EGFP positive (right). Scale bar: 100 µm. (E) EG cell lines established from E11.5 PGCs of transgenic mice without bFGF and SCF. Gonad cell suspensions were seeded onto MEFs, cultured with LIF and 300 nM 4OHT, and then passaged to tertiary cultures. Scale bar: 100 µm. (F) Mixed culture of wild-type and transgenic cells. Gonad cell suspensions of E11.5 wild-type and transgenic mice were cultured on Sl/Sl4-m220 feeder cells in the presence of LIF and 300 nM 4OHT, and passaged to tertiary cultures. All of the EG cell colonies were positive for EGFP (right). Scale bar: 250 µm.

View larger version (102K): [in this window] [in a new window]   Fig. 4. Multipotent differentiation capacities of EG cells established in AKT-MER transgenic mice. (A) Hematoxylin and Eosin (HE) staining of teratomas derived from EG cells established without bFGF. The cells differentiated into various tissues, including squamous epithelia (left), glands (middle) and cartilage (right, arrowhead). (B) HE staining of teratomas derived from EG cells established without bFGF and SCF. The teratomas were composed of various tissues, including cartilage (left), mucosal glands (middle) and muscle (right). Scale bar: 100 µm. (C) In vitro hematopoietic cell differentiation. EG cells established in the absence of bFGF (left), and the absence of bFGF and SCF (right), produced various hematopoietic cells in the OP9 differentiation system. Scale bar: 50 µm. (D) Chimeras derived from the transgenic EG cells established without bFGF. The contribution of the EG cells could be assessed by monitoring the EGFP fluorescence. In the E12.5 embryos (left and middle), EGFP fluorescence was distributed in the entire body. Right, newborn non-chimeric (middle) and chimeric mice (left, right). L, live; D, dead at birth. Left chimera survived to adult. (E) Chimeras derived from transgenic EG cells established without bFGF and SCF. Shown is the E12.5 chimera.

View larger version (60K): [in this window] [in a new window]   Fig. 5. The AKT downstream proteins and EG cell lines derived from p53-deficient mice. (A-C) Immunostaining with antibodies against phospho-GSK3 (A), MDM2 (B) and Ser20-phospho-p53 (C). The AKT-MER transgenic PGCs were cultured for 2 days in the presence of the indicated reagents. The cells were stained with antibodies against AKT downstream molecules (red) and germ cell markers SSEA-1 or PGC7/Stella (green). Yellow and white arrowheads indicate the stained and unstained PGCs, respectively. The percentage of positive cells is shown below each picture. The percentage of phospho-GSK3-positive PGCs (A) and of PGCs stained strongly with MDM2 antibody (B) was significantly higher in the 4OHT-treated PGCs, irrespective of bFGF treatment (*P<0.005, 2 test), than in wild type. The percentage of phospho-p53-positive PGCs cultured without 4OHT and bFGF was significantly higher than that of other groups (*P<0.005, 2 test; C). More than 300 PGCs were analyzed in each group. Scale bars: 25 µm. (D) EG cell lines derived from p53-deficient mice. EG cell lines were established in the presence (left) or absence (right) of bFGF. Scale bar: 100 µm.

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