Polypyrimidine tract-binding protein is essential for early mouse development and embryonic stem cell proliferation Masaki Shibayama*, Satona Ohno*, Takashi Osaka, Reiko Sakamoto, Akinori Tokunaga, Yuhki Nakatake, Mitsuharu Sato and Nobuaki Yoshida Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Japan Keywords cell cycle; embryonic stem cells; knockout mouse; polypyrimidine tract-binding protein; proliferation Correspondence N Yoshida, Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Fax: +81 5449 5455 Tel: +81 5449 5753 E-mail: nobuaki@ims.u-tokyo.ac.jp Polypyrimidine tract-binding protein (PTB) is a widely expressed RNAbinding protein with multiple roles in RNA processing, including the splicing of alternative exons, mRNA stability, mRNA localization, and internal ribosome entry site-dependent translation Although it has been reported that increased expression of PTB is correlated with cancer cell growth, the role of PTB in mammalian development is still unclear Here, we report that a homozygous mutation in the mouse Ptb gene causes embryonic lethality shortly after implantation We also established Ptb) ⁄ ) embryonic stem (ES) cell lines and found that these mutant cells exhibited severe defects in cell proliferation without aberrant differentiation in vitro or in vivo Furthermore, cell cycle analysis and a cell synchronization assay revealed that Ptb) ⁄ ) ES cells have a prolonged G2 ⁄ M phase Thus, our data indicate that PTB is essential for early mouse development and ES cell proliferation *These authors contributed equally to this work (Received 15 July 2009, revised 11 September 2009, accepted 15 September 2009) doi:10.1111/j.1742-4658.2009.07380.x Introduction Mouse embryonic stem (ES) cells are established from the inner cell mass (ICM) of blastocysts ES cells are defined by their ability to give rise to a variety of mature progeny while maintaining their capacity to self-renew Self-renewal is the process by which a stem cell divides to generate one or two daughter stem cells with developmental potentials that are indistinguishable from that of the mother cell This process is central to development, as well as to the maintenance of adult tissues in complex and long-lived organisms Self-renewal of ES cells is coordinated by multiple pathways, some of which are conserved among diverse types of stem cells, but others of which are restricted to certain cell types or tissues [1] In some of these pathways, alternatively spliced gene products have a variety of functions across multiple developmental stages [2] In addition, computational and experimental analyses have suggested that alternative splicing is important for ES cell self-renewal and differentiation [3] However, the mechanisms by which molecules that Abbreviations AP, alkaline phosphatase; E, embryonic day; EB, embryoid body; ES, embryonic stem; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICM, inner cell mass; IRES, internal ribosome entry site; LIF, leukemia inhibitory factor; PI, propidium iodide; PTB, polypyrimidine tract-binding protein; SCID, severe combined immunodeficiency; SD, standard deviation; SSEA-1, stage-specific embryonic antigen-1 6658 FEBS Journal 276 (2009) 6658–6668 ª 2009 The Authors Journal compilation ª 2009 FEBS M Shibayama et al regulate alternative splicing contribute to ES cell function are still elusive Polypyrimidine tract-binding protein (PTB; also known as PTBP1 ⁄ hnRNP I) is an alternative splicing regulator that is also widely expressed also in the early embryo [4,5] PTB regulates alternative exon inclusion in many genes, including Ptb itself [6,7] PTB has also been implicated in many aspects of mRNA regulation, including polyadenylation [8], stabilization [9,10], transcription [11], and localization [12,13] In addition, PTB is involved in internal ribosomal entry site (IRES)-dependent translation of cellular and viral genes [14,15] PTB has two paralogs, nPTB (also known as brPTB or PTBP2) and ROD1, which are expressed in a tissue-restricted manner nPTB is mostly expressed in neurons [16,17], and ROD1 is expressed in hematopoietic cells [18] Recently, it has been reported that increased expression of PTB is associated with ovarian tumor cell growth [19], and that PTB differentially affects cancer cell malignancy, depending on the cell line [20] In the context of development, PTB has been shown to be involved in germ cell differentiation in Drosophila melanogaster [4], and is essential for the development of Xenopus laevis [5] Although the importance of PTB for multiple biological processes has been reported, it is still unclear how PTB contributes to mammalian development and organogenesis To address these questions, we disrupted Ptb in mouse ES cells and generated Ptb knockout mice Homozygous mutation of Ptb resulted in embryonic lethality and revealed the importance of PTB in mouse development To elucidate the function of PTB in ES cells, we generated Ptb) ⁄ ) ES cells Although Ptb) ⁄ ) ES cells are viable, they form compact colonies and exhibit severe defects in cell proliferation without precocious differentiation Our data clearly demonstrate that PTB is essential for mouse development and ES cell proliferation Results Homozygous mutation of Ptb leads to embryonic lethality Previous reports have shown that Ptb is expressed in a wide variety of mouse tissues [16,21] and has multiple functions in somatic cells [6,8,10,12,15] However, the expression pattern and function of PTB in early development have not yet been elucidated To determine the role of PTB in mouse development, we generated Ptb-deficient mice through targeted gene disruption To introduce the null mutation for Ptb, we designed a targeting vector to replace a 1.9 kb region of Ptb PTB in development and ES cells on chromosome 10C1, including the promoter and transcriptional start site, with a neomycin resistance gene (Fig 1A; see detail in Doc S1) We introduced the targeting vector into E14.1 ES cells by electroporation, and screened G418-resistant clones for homologous recombination Southern blot analysis showed that seven of 240 clones were positive for homologous recombination To generate chimeric mice, we independently injected two heterozygous ES cell clones into C57BL ⁄ mouse blastocysts The chimeric mice derived from both clones successfully transmitted the mutated allele, and heterozygous mutant mice were produced by breeding Both male and female Ptb+ ⁄ ) mice were fertile, and showed no apparent defects To generate Ptb) ⁄ ) mice, we intercrossed heterozygous mutant mice, and analyzed the genotypes of the offspring by Southern blot and PCR Among the 16 neonatal mice examined, no homozygous mutants were observed (Table 1), indicating that Ptb) ⁄ ) embryos not survive to birth To determine the developmental stage of lethality, we genotyped embryos from embryonic day (E) 3.5 (blastocyst stage) to E10.5 As summarized in Table 1, no homozygous mutants were observed after E6.5, whereas the genotype ratio of embryos from E3.5 fitted the expected Mendelian ratio Thus, we deduced that homozygous mutation for Ptb leads to embryonic lethality shortly after implantation Characterization of Ptb–/– blastocysts To assess the protein expression of PTB in mouse early development, we performed immunohistochemical analysis on wild-type blastocysts We detected the immunoreactivity of PTB both in the ICM and in the trophectoderm (Fig 2A) In contrast, the expression of Oct3 ⁄ and Cdx-2 was restricted exclusively to the ICM or the trophectoderm (Fig 2A) In order to investigate the events surrounding implantation, we performed the blastocyst outgrowth assay We cultured the blastocysts for days and analyzed the genotypes by PCR Wild-type blastocysts exhibited normal outgrowth formations and were positive for alkaline phosphatase (AP) activity (Fig 2B) In contrast, although Ptb) ⁄ ) blastocysts were positive for AP activity, the growth rate of the ICM was reduced (Fig 2B) These results suggest that PTB is essential for embryonic development during the peri-implantation period Generation of the Ptb–/– ES cells The above data led us to analyze PTB function in ES cells, as these cells are derived from the ICM To FEBS Journal 276 (2009) 6658–6668 ª 2009 The Authors Journal compilation ª 2009 FEBS 6659 PTB in development and ES cells M Shibayama et al kb A A Wild-type locus (Chr 10C1) Sm Bg A Bg E Ss exon probe A Sm Targeting vector BH Sm BH BH A probe B BH Ss MC1 DT-A PGK Neo Targeting vector Probe A BH Ss A PGK Hyg MC1 DT-A wild-type: 9.1 kb 1st targeting: 26.0 kb 2nd targeting: 11.4 kb Probe B B Wild-type: 7.1 kb 1st targeting: 2.7 kb 2nd targeting: 9.4 kb Probe A C –/flox-hyg Probe B +/+ +/– –/flox-hyg +/– –/flox-hyg +/+ PGK Neo 9.4 kb 26.0 kb PGK Hyg exon –/flox 7.1 kb 11.4 kb Cre recombinase PGK Neo 9.1 kb Cre recombinase –/– PGK Neo 2.7 kb D Probe B –/flox –/– 7.1 kb exon 5.2 kb 2.7 kb PGK Neo Fig Gene targeting of mouse Ptb (A) Targeting strategy of Ptb The mutated Ptb allele was generated by homologous recombination (A) A, AflII, Bg, BglII; BH, BamHI; E, EcoRI; Sm, SmaI; Ss, Sse8387I (B) Southern blot analysis using the probes described in (A) Left panel: digested with AflII and detected by probe A Right panel: digested with BamHI and detected by probe B (C) Conditional disruption of Ptb in ES cells Ptb) ⁄ flox ES cells were generated by expression of Cre in Ptb) ⁄ flox-hyg ES cells Ptb) ⁄ ) ES cells were generated by infection of Ptb) ⁄ flox ES cells with a retroviral vector expressing Cre recombinase (D) Southern blot analysis of Ptb) ⁄ ) ES cells; digested with BamHI and detected by probe B Table Genotypes of offspring from Ptb+ ⁄ ) intercross The heterozygous mutant mice were intercrossed The genotypes of offspring were analyzed by Southern blot and PCR analysis Among 16 neonatal mice examined, no homozygous mutant was observed Stage +⁄+ +⁄) )⁄) Resorbed Total E3.5 E6.5 E8.5 E10.5 Newborn 11 14 13 24 26 17 12 0 0 – 13 18 – 43 15 53 48 16 6660 gain further insight into the function of PTB in ES cells, we first tried to establish Ptb) ⁄ ) ES cells from Ptb) ⁄ ) blastocysts; however, we could not obtain the Ptb) ⁄ ) ES cells (Table 2), probably owing to the cell proliferation defect Then, we attempted to disrupt both alleles of Ptb, using a conditional gene-targeting approach We constructed the second conditional targeting vector with a hygromycin resistance gene to mutate the wild-type allele and make heterozygous (Ptb) ⁄ flox-hyg) ES cells (Fig 1A; see detail in Doc S1) In this vector, we designed three loxP sequences to FEBS Journal 276 (2009) 6658–6668 ª 2009 The Authors Journal compilation ª 2009 FEBS M Shibayama et al PTB in development and ES cells A B –/– +/+ E SSEA-1 ICM +/+ PTB TG Cdx2 –/–1 Oct3/4 Relative gene expression C D +/+ +/– –/– –/– –/–2 PTB ** * 1.5 ** Fgf5 Gata4 Gata6 0.5 GAPDH + +/ –/– –/– Oct3/4 + +/ –/– –/– Sox2 + + +/ –/– –/– +/ –/– –/– Nanog Rex-1 Fig Characterization of blastocysts and Ptb) ⁄ ) ES cells (A) Immunostaining of PTB, Oct3 ⁄ and Cdx2 in wild-type blastocysts PTB is expressed in the ICM and trophectoderm (left column) Oct3 ⁄ (red) and Cdx-2 (green) indicate the ICM and the trophectoderm, respectively (right column) (B) In vitro outgrowth assay of blastocysts Intercrossed embryos at E3.5 were collected and cultured for days The morphology and AP activity of Ptb) ⁄ ) blastocysts were compared with those of wild-type cells Reduced proliferation of the ICM from Ptb) ⁄ ) blastocysts was observed TG, trophoblastic giant cells (C) Quantitative real-time PCR analysis comparing the expression of undifferentiated markers in wild-type cells and two Ptb) ⁄ ) ES cell clones Oct3 ⁄ 4, Sox2, Nanog and Rex-1 transcripts were normalized to Gapdh transcripts Mean values ± standard deviation (SD) were plotted from data obtained in at least three independent experiments *P > 0.05, **P > 0.005 (D) Northern blot analysis of differentiated marker expression Total RNA isolated from wild-type, heterozygous and Ptb) ⁄ ) cells was hybridized with radiolabeled cDNA probes Five micrograms of total RNA was loaded onto each lane (E) SSEA-1 expression in wild-type and Ptb) ⁄ ) ES cells ES cells were cultured on a feeder layer The expression of SSEA-1 was maintained in both wild-type cells and the two Ptb) ⁄ ) ES cell clones GAPDH, glyceraldehyde-3-phosphate dehydrogenase Scale bar: 100 lm flank the hygromycin resistance gene cassette and the 1.9 kb genomic fragment containing the promoter and transcriptional start site of Ptb (Fig 1A) We introduced the vector into Ptb+ ⁄ ) ES cells, which we Table Genotypes of established ES cell lines from blastocysts Fifty-one blastosysts from heterozygous intercrossing were used for ES cell derivation After 2–3 weeks of culture, ES cell lines were established from 14 blastosysts All of the ES cell lines were positive for AP activity Whereas heterozygous or wild-type cell lines were obtained with the expected Mendelian ratio, no Ptb) ⁄ ) ES cell line was found Genotype +⁄+ ES cell line +⁄) )⁄) Total 14 generated using the first targeting vector We screened the hygromycin-resistant colonies for homologous recombination, and obtained positive clones, which were mutated at the wild-type allele (Ptb) ⁄ flox-hyg; Fig 1B) To generate Ptb) ⁄ ) ES cells, we introduced a Cre expression vector into Ptb) ⁄ flox-hyg cells by electroporation (Fig 1C) Although we obtained Ptb) ⁄ flox cells, we failed to establish Ptb) ⁄ ) ES cells Then, we expressed Cre by retrovirus infection into Ptb) ⁄ flox cells (Fig 1C) To identify Ptb) ⁄ ) ES cells, we screened those cells by PCR and confirmed their genotypes by Southern blot analysis, and we successfully identified two independent Ptb) ⁄ ) ES cell clones () ⁄ )1 and ) ⁄ )2) (Fig 1D) The expression of Ptb mRNA and protein was completely abolished in both Ptb) ⁄ ) ES cells (Fig 5A and Fig S2) FEBS Journal 276 (2009) 6658–6668 ª 2009 The Authors Journal compilation ª 2009 FEBS 6661 PTB in development and ES cells M Shibayama et al Ptb–/– ES cells maintain the undifferentiated state To address the expression profile of the undifferentiated markers between wild-type and Ptb) ⁄ ) ES cells, the relative abundance of selected mRNAs was determined by quantitative real-time PCR analysis The expression of Nanog was slightly decreased in both Ptb) ⁄ ) ES cell clones, and Rex-1 expression was reduced in one of the Ptb) ⁄ ) ES cell clones () ⁄ )2) as compared with that of wild-type ES cells (Fig 2C) Although it has been reported that the expression of undifferentiated marker genes, such as Oct3 ⁄ and Sox2, is decreased in Nanog-deficient ES cells [22], the A expression of Oct3 ⁄ and Sox2 mRNA was maintained and not different between the two Ptb) ⁄ ) ES cell clones (Fig 2C) Furthermore, both Ptb) ⁄ ) ES cell clones also expressed another ES cell marker, stage-specific embryonic antigen-1 (SSEA-1) (Fig 2E), and were positive for AP activity (Fig 3A) In contrast, the expression of SSEA-1 was not detected in the differentiated ES cells (Fig S1) On the other hand, the northern blotting and quantitative real-time PCR analysis also showed that the expression of differentiation marker genes such as fgf5, gata4 and gata6 was not increased in wild-type cells or either of the Ptb) ⁄ ) ES cell clones (Figs 2D and 4A) Taken together, these results indicate that B D PI TUNEL +/+ Cell number ( x 106) +/+ +/+ +/– –/– +/– –/–1 C Day –/–1 Fold increase 100 –/–2 –/–2 50 IB:anti-PTB +/+ +/– –/– –/– –/– –/– –/– –/– E +/+ (Serum free) Ptb Tg vector Relative viable cells 1.4 +/+ 1.2 0.8 –/–1 0.6 0.4 0.2 0 Days Fig Reduced proliferation of Ptb) ⁄ ) ES cells (A) AP staining of wild-type and Ptb) ⁄ ) ES cells The AP activities were positive in both wild-type cells and the two Ptb) ⁄ ) ES cell clones Scale bar: 100 lm (B) Cell proliferation assay Cells (5 · 104) were seeded (d0) and counted every day for days of culture The proliferation of Ptb) ⁄ ) ES cells was reduced as compared with wild-type cells (C) Impairment of cell proliferation seen in the Ptb) ⁄ ) ES cells was rescued by ectopic expression of PTB Ptb) ⁄ ) ES cell clones were stably transfected with a PTB expression vector or control plasmid, and subjected to a cell proliferation assay as in (B) Bars indicate fold increase in cell number after days of cell culture The amount of ectopically expressed PTB was comparable to that expressed by heterozygous ES cells (lower panel) The concentration of lysates was quantified, and the same volume was loaded into each lane Tg, transgene (D) Apoptosis assay Left, bright field; middle, PI staining; right, fluorescence-labeled DNA fragmented by terminal deoxynucleotidyl transferase TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling Wild-type ES cells cultured under serum-free conditions was used as a positive control for the apoptosis assay Scale bar: 200 lm (E) Proportion of viable cells Cell viability was calculated as the ratio of the number of Trypan blue-staining-negative cells to that of total cells Cells (3 · 105) were seeded on growth medium and counted every day for days of culture The circles indicate the values for wild-type cells and the triangles indicate those for Ptb) ⁄ ) ES cells Mean values ± SD were plotted from data obtained in experiments conducted in triplicate *P > 0.05 6662 FEBS Journal 276 (2009) 6658–6668 ª 2009 The Authors Journal compilation ª 2009 FEBS M Shibayama et al )⁄) Although Ptb ES cell clones were viable and formed typical oval-shaped compact colonies on feeder layers, Ptb) ⁄ ) ES cell colonies were smaller than control cell colonies (Fig 3A) A cell proliferation assay showed that wild-type and parental Ptb+ ⁄ ) ES cells were able to expand more than 60-fold after days of culture, whereas both Ptb) ⁄ ) ES cell clones showed only a fivefold to seven-fold increase in the same period (Fig 3B) To confirm whether the reduced proliferation rate of Ptb) ⁄ ) ES cells was due to loss of PTB expression, we introduced the PTB expression vector into Ptb) ⁄ ) ES cells (Fig 3C) The proliferation defect seen in Ptb) ⁄ ) ES cells was recovered by PTB re-expression (Fig 3C), suggesting that the defect in cell proliferation of Ptb) ⁄ ) ES cells was due to the loss of PTB expression As we observed no signs of apoptosis (Fig 3D) or massive cell death (Fig 3E) in Ptb) ⁄ ) ES cell cultures, the small size of Ptb) ⁄ ) ES cell colonies and the results of our proliferation assay indicate a reduced proliferation rate in Ptb) ⁄ ) ES cells To further investigate the proliferative ability of Ptb) ⁄ ) ES cells, we assessed their teratoma formation ability in vivo We transplanted wild-type or Ptb) ⁄ ) ES cells under the kidney capsules of five severe combined immunodeficiency (SCID) mice, and examined the kidneys weeks after transplantation (Fig 4B) The wet weight of teratomas resulting from transplantation with Ptb) ⁄ ) ES cells after weeks was more than 20-fold reduced as compared with wild-type teratomas (Fig 4C) To determine whether the teratoma formation defect of Ptb) ⁄ ) ES cells is due to loss of pluripotency, we performed embryoid body (EB) formation assay and quantified the expression of differentiation marker genes by quantitative real-time PCR (Fig 4A) The EBs were formed by suspension culture of ES cells for days without leukemia inhibitory factor (LIF) The quantitative real-time PCR analysis revealed that differentiation markers such as Fgf5, Gata4 and Gata6 were expressed in EBs from Ptb) ⁄ ) ES cells, as well as wild-type ES cells (Fig 4A) These results indicate that the defect of teratoma formation from Ptb) ⁄ ) ES cells is not due to the loss of pluripotency Interestingly, the expression levels of differentiation markers in EBs from Ptb) ⁄ ) ES cells were higher than those in wildtype cells, indicating that Ptb) ⁄ ) ES cells may have a greater tendency to differentiate than wild-type ES 100 * 80 * 60 40 20 + + +/ –/– –/– +/ –/– ES EB Fgf5 B –/– + + + + +/ –/– –/– +/ –/– +/ –/– –/– +/ –/– ES EB Gata4 C +/+ mm ES EB Gata6 3.5 Wet weight (g) Ptb) ⁄ ) ES cells exhibit reduced cell proliferation A Relative gene expression although the expression of a part of ES cell-specific markers was reduced in Ptb) ⁄ ) ES cells, both Ptb) ⁄ ) ES cell clones still remained in undifferentiated state and did not lead to precocious differentiation PTB in development and ES cells 2.5 1.5 0.5 +/+ –/– Fig Ptb) ⁄ ) ES cells have a severe defect in cell proliferation in vivo and in vivo (A) Quantitative real-time PCR analysis comparing the expression of differentiated markers in wild-type and Ptb) ⁄ ) ES cells and EBs from wild-type and Ptb) ⁄ ) ES cells () ⁄ )1) Fgf5, Gata4 and Gata6 transcripts were normalized to Gapdh transcripts Mean values ± SD were plotted from data obtained in at least three independent experiments *P > 0.05 (B) Teratoma formation by ES cell transplantation Wild-type or Ptb) ⁄ ) ES cells were transplanted under the kidney capsules of SCID mice Three weeks after transplantation, teratoma formation was examined In four of five mice transplanted with wild-type ES cells, a teratoma formed around the kidney (right) However, no teratoma formation was observed in the five mice transplanted with Ptb) ⁄ ) ES cells (left) Scale bar: mm (C) Wet weight of teratomas The average wet weight of teratomas resulting from transplantation with wild-type ES cells was approximately 2.3 g Mean values ± SD were plotted from data obtained in experiments conducted in triplicate *P > 0.01 cells Collectively, our data demonstrate that PTB is one of the critical factors for proliferation but not pluripotency of ES cells both in vitro and in vivo FEBS Journal 276 (2009) 6658–6668 ª 2009 The Authors Journal compilation ª 2009 FEBS 6663 PTB in development and ES cells M Shibayama et al Ptb–/– ES cells have prolonged G2/M progression To further characterize the reduced proliferation phenotype seen in Ptb) ⁄ ) ES cells, we measured the expression of several well-known cell cycle regulators by western blot analysis (Fig 5A) Although it has been reported that PTB modulates the G1 to S transition through enhancement of IRES-dependent translation of p27kip1 in differentiated cells such as 293T cells [23], the protein level of p27kip1 in Ptb) ⁄ ) ES cells was not different from that in wild-type ES cells (Fig 5A) Moreover, no alterations in cyclin A, B or E protein expression were found in Ptb) ⁄ ) ES cells (Fig 5A) These results indicate that the cause of the proliferation defect in Ptb) ⁄ ) ES cells is not aberrant expression of these cell cycle regulators To further investigate the mechanism of the cell proliferation defect seen in Ptb) ⁄ ) ES cells, we performed cell cycle analysis We fixed and stained cells with propidium iodide (PI), after which we analyzed the DNA content by flow cytometry (Fig 5B) The peak of the cell population mapped in the G2 ⁄ M phase was higher in Ptb) ⁄ ) ES cells than in wild-type ES cells (Fig 5B) A B This result suggests that the cause of the proliferation defect in Ptb) ⁄ ) ES cells may be G2 ⁄ M phase delay We next analyzed cell cycle progression in Ptb) ⁄ ) ES cells by arresting the cells in the early S phase with a double thymidine block We released cells from the block and fixed them at the time points indicated in Fig 5C, and then analyzed the DNA contents of the cells by flow cytometry Up until h after the release, the DNA content patterns were essentially the same in Ptb) ⁄ ) and wild-type ES cells, and cells were at the end of the S phase in this period (Fig 5C, shaded in gray) These results indicate that progression through the S phase is not affected by PTB deficiency However, the number of cells returning to the G1 phase through the G2 ⁄ M phase was smaller in Ptb) ⁄ ) ES cells than in control cells, as seen at h after release (Fig 5C, indicated by arrows) As the pattern of DNA contents at h after release in Ptb) ⁄ ) ES cells was the same as that after h in control cells, we estimated the delay in G2 ⁄ M progression in Ptb) ⁄ ) ES cells to be approximately h Taking these data together, we conclude that the proliferation defect in Ptb) ⁄ ) ES cells is a result of delayed G2 ⁄ M progression C Fig G2 ⁄ M progression is delayed in Ptb) ⁄ ) ES cells (A) Expression of cell cyclerelated proteins in Ptb) ⁄ ) ES cells Expression of p27, cyclin A, cyclin B and cyclin E was examined by western blotting Whole cell extracts from wild-type, heterozygous and Ptb) ⁄ ) ES cells were subjected to SDS ⁄ PAGE No significant difference was observed in Ptb) ⁄ ) ES cells (B) Cell cycle analysis of asynchronous Ptb) ⁄ ) ES cell populations by flow cytometry Cells were fixed in ethanol and stained by PI The percentage of cells in the G2 ⁄ M stage is described in the histograms %G1: + ⁄ +, 10.0%; ) ⁄ )1, 8.99%; ) ⁄ )2, 9.76% %S: + ⁄ +, 67.9%; ) ⁄ ), 64.2%; ) ⁄ )2, 65.9% The experiment was independently repeated at least three times (C) Cell synchronization assay for cell cycle progression analysis Wild-type and Ptb) ⁄ ) ES cells were synchronized by a double thymidine block Cells were fixed at the indicated time points after release, and the DNA content of the cells was analyzed by flow cytometry Arrows indicate differences in G1 peak appearance between wild-type and Ptb) ⁄ ) ES cells 6664 FEBS Journal 276 (2009) 6658–6668 ª 2009 The Authors Journal compilation ª 2009 FEBS M Shibayama et al Discussion We have shown that PTB, which has multiple functions in RNA metabolism, is an essential factor in mouse early development and ES cell proliferation To assess the function of PTB in vivo and in vitro, we used a strategy in which Ptb was mutated by homologous recombination, and determined that the Ptb knockout mice exhibited embryonic lethality shortly after implantation (Table 1) We then established two Ptb) ⁄ ) ES cell lines, and found that Ptb) ⁄ ) ES cells showed severe defects in cell proliferation in vivo and in vitro (Figs 3B and 4B) As Ptb) ⁄ ) ES cells exhibit a low proliferation rate, Ptb) ⁄ ) ES cells may not be established from Ptb) ⁄ ) blastocysts or Cre-transfected Ptb) ⁄ flox-hygro ES cells Lower proliferation rates are also found in sall4-disrupted, klf5-disrupted, HDAC1-disrupted, ronin-disrupted and dicer-disrupted ES cells relative to wild-type ES cells, and mice with knockout of these genes also show embryonic lethality at the peri-implantation stage [24–28], a phenotype similar to that of the Ptb knockout mice Although the phenotypes of ES cells with disruption of these genes differ, these reports suggest that a lower ES cell proliferation rate can cause critical defects in embryonic development As PTB is expressed in both the ICM and the trophectoderm, we could not exclude the possibility of a failure of implantation due to defective trophectoderm development, as in the case of the klf5 knockout mice [25] In klf5) ⁄ ) ES cells, expression of differentiation-related genes and spontaneous differentiation are increased [25] However, these phenotypes are not observed in Ptb) ⁄ ) ES cells Furthermore, the expression of Oct3 ⁄ was not disturbed in Ptb) ⁄ ) ES cells, and this is different from what is seen in sall4) ⁄ ) ES cells [24] These reports suggest that regulation of proliferation occurs through more than one mechanism in ES cells One likely reason for the embryonic lethality of the Ptb knockout mice is the prolonged G2 ⁄ M progression seen in Ptb) ⁄ ) ES cells (Fig 5B,C) As proposed in a recent review [29], mitosis is a key process in which transcriptional programs are altered From our results showing that Ptb) ⁄ ) ES cells have a prolonged G2 ⁄ M phase and Ptb knockout mice exhibit embryonic lethality, it appears that irregular control of the mitotic phase may affect nuclear reorganization processes, resulting in loss of control of transcriptional programs This difference in developmental regulation may also apply to the mechanisms of promiscuous gene expression and other phenotypes seen in cancer cells In ovarian cancer, a high level of expression of PTB is correlated with tumor cell growth and malignancy [19,20] This may be due to disruption of the gene expression program in tumor cells resulting from PTB in development and ES cells augmented PTB expression Taken together, these data suggest that PTB is a key factor in switching of cell identity through mitotic phase modulation PTB is a multifunctional protein that is involved in transcription, polyadenylation, alternative splicing, and IRES-dependent translation, and these steps are all known to be targets for mitotic inhibition [30,31] The regulatory mechanism of the ES cell cycle is still unclear We are currently investigating whether PTB is one of the important regulators for the G2 ⁄ M phase in ES cells Cell proliferation and differentiation are highly coordinated processes during development, and it is well known that, in many systems, terminal differentiation is coupled with growth arrest The low proliferation rate may be responsible for the rapid differentiation potential of Ptb) ⁄ ) ES cells, and result in higher expression levels of differentiated marker genes in EBs from Ptb) ⁄ ) ES cells than in EBs from wild-type cells (Fig 4A) The expression of the undifferentiated stem cell marker Nanog is downregulated in both Ptb) ⁄ ) ES cell clones (Fig 2C) We observed that recombinant PTB protein can bind to a pyrimidine-rich sequence in the Nanog promoter region (Y Nakatake, unpublished data) These data suggest that PTB may partially regulate the expression of Nanog The difference in Nanog expression between the two Ptb) ⁄ ) ES cell clones may be due to the effect of factor(s) other than PTB As the expression of Rex-1 is regulated by Nanog [32,33], the reduction of Rex-1 expression in Ptb) ⁄ ) ES cells () ⁄ )2) may be caused by downregulation of Nanog In the other clone () ⁄ )1), the expression level of Nanog may be enough to activate Rex-1 expression Although the expression levels of Nanog and Rex-1 are different between the two Ptb) ⁄ ) ES cell clones, we did not observe any differences in phenotypes such as proliferation (Fig 3B), apoptosis (Fig 3D), or undifferentiated state (Figs 2E and 3A) Furthermore, in Ptb) ⁄ ) ES cells, we did not observe any spontaneous differentiation (Figs 2D and 4A) or downregulation of Oct3 ⁄ (Fig 2C), as is seen in Nanog-deficient ES cells [22] Collectively, these data suggest that the phenotypes resulting from the absence of PTB are due to a distinct mechanism that is independent of Nanog and Oct3 ⁄ PTB regulates nonsense-mediated decay of transcripts of nPTB, which is one paralog of PTB [34] We investigated whether the expression of nPTB was increased in Ptb) ⁄ ) ES cells (Fig S2) The level of nPTB in Ptb) ⁄ ) ES cells was higher than in wild-type ES cells Although it has been reported that PTB and nPTB have functional overlap [35] in HeLa cells, the increase of nPTB expression did not rescue the proliferation defect in ES cells Our study has revealed the importance of PTB in cell proliferation Questions that still need to be answered FEBS Journal 276 (2009) 6658–6668 ª 2009 The Authors Journal compilation ª 2009 FEBS 6665 PTB in development and ES cells M Shibayama et al are what the identity is of the target protein regulated by PTB in the mitotic phase and how this target protein modulates mitosis and cell proliferation The answers to these questions will provide novel insights into gene regulation through mitosis Another interesting approach would be to clarify the significance of PTB in cells without a mitotic cycle Heart and brain tissues may be interesting in this respect, as they express PTB [16,21] but not engage in massive cell growth These experiments are now possible, owing to our establishment of conditional targeting of Ptb in mice The molecular mechanisms of PTB regulation of early mouse development and ES cell proliferation are important questions that are worthy of further investigation Experimental procedures (Invitrogen, Carlsbad, CA, USA) Linearized PTB expression vector or pBPCAGGS was then transfected into cells with Lipofectamine2000 (Invitrogen), and the cells were selected in the presence of lgỈmL)1 blasticidin (InvivoGen, San Diego, CA, USA) for days Northern blotting Total RNA was isolated by ultracentrifugation [36] or extracted using sepasol RNA I (Nacalai Tesque, Kyoto, Japan) Agarose gel electrophoresis and blotting were performed as previously reported [37] Hybridization and washing of the blotted filter were performed according to previously described methods [38] Probes for Fgf5, Gata4 and Gata6 were obtained by PCR amplification Primer sequences are described in Table S1 cDNA templates for probes were synthesized by SuperScriptII ⁄ III (Invitrogen) according to the manufacturer’s instructions Cell culture ES cells were cultured in DMEM (Nissui, Tokyo, Japan) supplemented with LIF, 15% fetal bovine serum, 100 nm 2mercaptoethanol, 0.06% l-glutamine, and glucose (to a final concentration of 4500 mgỈL)1) Mouse embryonic fibroblasts were maintained in DMEM supplemented with 10% fetal bovine serum and 0.06% l-glutamine Hygromycin-resistant MEFs were prepared from mice generously provided by Y Iwakura (IMSUT, Japan) Proliferation assay, apoptosis assay, and AP staining For the proliferation assay, · 105 cells were seeded in growth medium and counted every day over days of culture Viable and total cells were counted with and without Trypan blue solution The value of relative viable cells was calculated as the ratio of the number of Trypan blue-negative cells to that of total cells The apoptosis assay was performed using an ApopTag Fluorescein Direct In Situ Apoptosis Detection Kit (Chemicon), following the manufacturer’s instructions AP staining was performed using an AP leukocyte kit (Sigma-Aldrich, St Louis, MO, USA), following the manufacturer’s instructions PTB expression vector and plasmid transfection The coding sequence for PTB was obtained by PCR amplification using relevant primers (Table S1) The resulting cDNA fragment was digested with HindIII and SlaI, and then subcloned into pBluescript II (Stratagene, La Jolla, CA, USA) and sequenced For the PTB expression vector, the cDNA was ligated into pBPCAGGS, in which the pHPCAGGS hygromycin resistance gene cassette (kindly provided by H Niwa, RIKEN, Japan) was replaced with a blasticidin resistance gene cassette from pcDNA6 ⁄ TR 6666 Quantitative real-Time PCR analysis For the RT-PCR analysis, first-strand cDNA was synthesized from lg of total RNA that had been treated with DNase I in 10 lL of reaction mixture using the High Capacity RNA-to-cDNA Kit (ABI, Foster City, CA, USA) The quantitative real-time PCR reaction was performed with a Fast SYBR Green Master Mix (ABI) and analyzed on a StepOnePlus (ABI) Relative gene expression was calculated using the standard curve method The sequences of primers for quantitative real-time PCR are listed in Table S1 Antibodies and immunodetection Rabbit anti-Oct3 ⁄ (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Oct3 ⁄ [39], mouse anti-PTB (Zymed, Invitrogen), rabbit anti-SSEA-1 (Chemicon, Millipore, Billerica, MA, USA), mouse anti-p27kip1 (BD Pharmingen, Franklin Lakes, NJ, USA), rabbit anti-cyclin A (Santa Cruz Biotechnology) and rabbit anti-cyclin E (Santa Cruz Biotechnology) sera were used for immunodetection For immunofluorescent staining, Alexa Fluor 488 anti-rabbit IgG (Molecular Probes, Invitrogen) and Alexa Fluor 562 antimouse IgG (Molecular Probes) were used as secondary antibodies For western blotting, horseradish peroxidase-linked anti-mouse IgG and anti-rabbit IgG (GE Healthcare, Chalfont St Giles, UK) were used Immunoreactivity was detected using an enhanced chemiluminescence kit (GE Healthcare) and X-ray film (Fuji Film, Kanagawa, Japan) Cell cycle analysis A double thymidine block was performed as follows Thymidine (MP Biomedicals, Illkirch, France) was added to each ES cell culture to a final concentration of mm After FEBS Journal 276 (2009) 6658–6668 ª 2009 The Authors Journal compilation ª 2009 FEBS M Shibayama et al 16 h, the cells were washed twice with NaCl ⁄ Pi and released for h in growth medium A second block was initiated by adding thymidine to a concentration of mm and was maintained for 16 h Cells were washed twice with NaCl ⁄ Pi, released in fresh growth medium for the indicated periods of time, and then fixed in cold 70% ethanol Fixed cells from the double thymidine block were treated with mgỈmL)1 RNaseA (Sigma, St Louis, MO, USA) and 50 lgỈmL)1 PI (Nacalai Tesque) for 30 at room temperature Cell cycle analysis was carried out using a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) and flowjo software (TreeStar, Ashland, OR, USA) Mice and teratoma formation C57BL ⁄ 6J mice and MCH:ICR mice were purchased from CLEA Japan (Tokyo, Japan) All of the mice were maintained under specific pathogen-free conditions in the animal facility of the IMSUT, the University of Tokyo For teratoma formation, wild-type or Ptb) ⁄ ) ES cells were suspended in NaCl ⁄ Pi and transplanted (3 · 105 cells per kidney) under the kidney capsules of adult male C.B-17 ⁄ Icr scid Jcl mice (CLEA Japan) Three weeks after transplantation, the kidneys were collected and examined All of the work with mice conformed to guidelines approved by the Institutional Animal Care and Use Committee of the University of Tokyo PTB in development and ES cells 10 11 Acknowledgements We thank R Kuhn for providing us with E14.1 ES ă cells, H Niwa for the pHPCAGGS plasmid and rabbit anti-Oct3 ⁄ serum, and Y Iwakura for hygromycinresistant mouse embryonic fibroblasts This research was supported by a Research Grant (2000–2004, to N Yoshida) for the 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Expression of nPTB in Ptb) ⁄ ) ES cells Doc S1 Construction of targeting vectors Table S1 List of primer sequences This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 276 (2009) 6658–6668 ª 2009 The Authors Journal compilation ª 2009 FEBS ... they form compact colonies and exhibit severe defects in cell proliferation without precocious differentiation Our data clearly demonstrate that PTB is essential for mouse development and ES cell. .. PTB in development and ES cells M Shibayama et al are what the identity is of the target protein regulated by PTB in the mitotic phase and how this target protein modulates mitosis and cell proliferation. .. causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development Development 133, 3005–3013 Ema M,