In the adult mammalian testis, spermatogenic differentiation starts from a minute population of spermatogonial stem cells (SSCs). SSCs are generated after birth from the fetal gonocytes, themselves derived from the primordial germ cells (PGCs), which are specified during the first days after implantation. Transcriptome profiling of purified preparations evidenced the preferential accumulation in SSCs of transcripts of PU.1(Sfpi1), a regulatory gene previously identified in hematopoietic progenitors. In situ immunolabeling and RNA determination showed a complex pattern of expression in the adult testis, first in SSCs and early spermatogonia followed by de novo expression in pachytene spermatocytes. Spermatogenesis in a null mutant (PU.1G/G) was arrested at the prenatal stage, with reduced numbers of gonocytes owing to a defect in proliferation already noticeable at E12.5. Transcripts of several germinal markers, including vasa (Mvh, Ddx4), Oct4 (Pou5f1), Dazl and Taf4b, were detected, whereas stella (PGC7,Dppa3) was not. Germ cells of PU.1G/G newborn testes grafted in nude mice did not initiate the postnatal replicative stage, whereas grafts of their wild-type littermates underwent complete spermatogenesis. During embryonic development, PU.1 transcription was initiated as early as the blastocyst stage, with a generalized expression at E6.5 in the embryonic ectoderm. PU.1 therefore appears to play a determinant role in at least two distinct lineages and, given its wide range of expression,possibly in other stem cells.

Male germinal differentiation in mammals comprises two distinct periods delimited by birth (reviewed by Hogan et al., 1994). During embryonic development, the primordial germ cells (PGCs) specified in the epiblast at embryonic day (E) 7.5 divide and colonize the gonadal ridge, where they become associated with somatic cells in the cord structures characteristic of the fetal testis. At the end of prenatal development, the germ cells (gonocytes) stop dividing. The spermatogonial stem cells (SSCs), which resume proliferation during the first days after birth(de Rooij and Grootegoed,1998), were functionally defined by their ability to reconstitute a functional seminiferous epithelium after transplantation into a sterile host(Brinster, 2002; Brinster and Zimmermann, 1994). The cyclic process of spermatogenesis initiated from SSCs will thereafter be maintained throughout the whole of adult life.

The two critical turning points in this process are the specification of PGCs in the early embryo and the appearance of dividing SSCs shortly after birth. Genes expressed at the earliest stages of specification of the germ line have been identified recently. They include fragilis (Ifitm3)(Saitou et al., 2002; Tanaka et al., 2005), stella(PGC7, Dppa3) (Bortvin et al.,2004; Nakamura et al.,2007; Payer et al.,2003; Saitou et al.,2002; Sato et al.,2002) and Blimp1 (Prdm1)(Ohinata et al., 2005; Saitou et al., 2005; Vincent et al., 2005). PGCs appear first in the proximal epiblast, in a region in which fragilis is upregulated at E6.5. This restricted PGC population (approximately 40 cells)expresses Blimp1 (E7.25) and thereafter (E7.5) stella. After birth,only a limited number of genes crucial for establishment and self-renewal of SSCs have been identified. Requirement for the Ret receptor was postulated based on the requirement for its ligand GDNF(Meng et al., 2000), and a partial requirement for Bcl6b was described recently(Oatley et al., 2006).

Our starting point was the establishment of a transcriptome profile of purified preparations of SSCs, on the assumption that crucial genes would appear among those expressed at elevated levels in the stem cells. Work on adult SSCs is made difficult by their small number. We previously reported an efficient procedure, in which a truncated version of the Stra8promoter is used to express in SSCs a neutral heterologous surface protein including two domains of the human CD4 antigen (huCD4)(Giuili et al., 2002). Once fractionated by immunomagnetic sorting, the huCD4-positive fraction is homogeneous for the expression of known SSC markers and highly efficient in the colonization of a sterile recipient. Hybridization on DNA arrays provided a shortlist of transcripts upregulated in the purified fraction. Interestingly, the list included PU.1 (Sfpi1), a member of the Ets family, which has been extensively studied for its important regulatory functions in the renewal of progenitor cells and in early differentiation, so far exclusively in the various lineages of hematopoietic differentiation (reviewed by Lloberas et al., 1999; Metcalf et al.,2006). The availability of a null mutation generated by insertion of the green fluorescent protein (GFP) reporter gene in the first exon(Back et al., 2004), allowed us to demonstrate here a strict requirement for PU.1 in the fetal differentiation of the male germ line and to show promoter activity starting at the earliest embryonic stages.

### Cell culture

129Sv ES cells were grown at 37°C, 5% CO2, 95% humidity in dishes coated with a feeder layer of mitotically inactivated primary mouse embryonic fibroblasts. Cells were kept in Dulbecco's modified Eagle's medium supplemented with 15% FCS, 100 IU/ml penicillin/streptomycin, 100 IU/ml non-essential amino acids, 0.1 mM 2-mercaptoethanol and 1000 IU/ml LIF.

### Transgenic mice

A truncated version of the mouse Stra8 promoter was used as described previously (Giuili et al.,2002) to direct expression to the surface of SSCs of a fusion protein including two domains of the human CD4 protein (Stra8-CD4HAglo mice). Progeny were genotyped by PCR amplification of tail DNA using primers hCD4F and hCD4R (Table 1). PU.1G/+ and PU.1G/G mice(Back et al., 2004) were genotyped by PCR with primers VP102 (wild-type exon 1), VP104 (mutated exon 1)and WF189 (intron 1) (Table 1). Amplified fragments were 650 bp for the PU.1+ allele and 900 bp for PU.1G. Families were maintained in the C57BL/6×DBA/2 F1 genetic background. Investigations were conducted in accordance with French and European rules for the care and use of laboratory animals.

Table 1.

Oligonucleotide primers

Amplified sequenceForward (F)Reverse (R)
PU.1+ (genomic) GGCCTACAGGAAGAGCAAGCCTGGCA ATTCGTTACAAATGCAAGCTAAA
PU.1G (genomic) AGGGGCGGTGAGGGAAAACCCTTCCA ATTCGTTACAAATGCAAGCTAAA
PU.1 (RNA) TGGGAGGGTTTTCCCTCACC TGCTGTCCTTCATGTCGCCG
huCD4 CCTCAGTGCAATGTAGGAGTC CTCATCAGATGCATATTCTGC
Stra8 TCACAGCCTCAAAGTGGCAGG GCAACAGAGTGGAGGAG
Gapdh CACCACCAACTGCTTAGCC CGGATACATTGGGGGTAGG
Kit CAACAGCAATGGCCTCACGAGT GTGGTCACCTTTGCTCTGCTC
Sycp1 ATGGAGAAGCAAAAGCCCTTC ATGTTTTCCCGACTGGACATTG
Prm1 GGCCAGATACCGATGC CCTTAGCAGGCTCCTG
β-actin AAGGCCAACCGTGAAAAGAT GTGGTACGACCAGAGGCATAC
Cd81 GGGCATCTACATTCTCATTGC GTATGGTGGTAGTCAGTGTGG
Cd97 CTCGGAGAAGGAATTGATCAC CTTCTGAGTCTCCCATCTGTG
Nptn GAATTGTCACCAGTGAAGAGG CATCATAGCATCCTGCCCTTC
Fos CAGCCTTTCCTACTACCATTC CTGACACGGTCTTCACCATTC
Cdc42 GGCAAGAGGATTATGACAGAC CAGCAGTCTCTGGAGTAATAG
Cd14 GGAAGCCAGAGAACACCATCG GCAGGGCTCCGAATAGAATCC
Lyn GAGAGAAGGCTTCATCCCCAG GTCTTCACAGCCACCTTTGTG
Nek7 GAGCATCCTGTCTCTTGGATG GACTCTTCGAGAATGCATGTG
stella AAAGCTAACCCTAAACCCCGG TGGCTCACTGTCCCGTTCA
vasa CCAAAAGTGACATATATACCC TTGGTTGATCAGTTCTCGAG
Plzf CCACCTTCGCTCACATACAG TGTGGCTCTTGAGTGTGCTC
Taf4b CAGACAGCCAAAGTCAAGCA TGGAGGTCACATGATTCACAC
Oct4 TGAGGCTACAGGACACCTTTC GTGCCAAAGTGGGGACCT
Dazl GCTGATATTTTGCCCAATGAA ATGCTTCGGTCCACAGACTT
Amplified sequenceForward (F)Reverse (R)
PU.1+ (genomic) GGCCTACAGGAAGAGCAAGCCTGGCA ATTCGTTACAAATGCAAGCTAAA
PU.1G (genomic) AGGGGCGGTGAGGGAAAACCCTTCCA ATTCGTTACAAATGCAAGCTAAA
PU.1 (RNA) TGGGAGGGTTTTCCCTCACC TGCTGTCCTTCATGTCGCCG
huCD4 CCTCAGTGCAATGTAGGAGTC CTCATCAGATGCATATTCTGC
Stra8 TCACAGCCTCAAAGTGGCAGG GCAACAGAGTGGAGGAG
Gapdh CACCACCAACTGCTTAGCC CGGATACATTGGGGGTAGG
Kit CAACAGCAATGGCCTCACGAGT GTGGTCACCTTTGCTCTGCTC
Sycp1 ATGGAGAAGCAAAAGCCCTTC ATGTTTTCCCGACTGGACATTG
Prm1 GGCCAGATACCGATGC CCTTAGCAGGCTCCTG
β-actin AAGGCCAACCGTGAAAAGAT GTGGTACGACCAGAGGCATAC
Cd81 GGGCATCTACATTCTCATTGC GTATGGTGGTAGTCAGTGTGG
Cd97 CTCGGAGAAGGAATTGATCAC CTTCTGAGTCTCCCATCTGTG
Nptn GAATTGTCACCAGTGAAGAGG CATCATAGCATCCTGCCCTTC
Fos CAGCCTTTCCTACTACCATTC CTGACACGGTCTTCACCATTC
Cdc42 GGCAAGAGGATTATGACAGAC CAGCAGTCTCTGGAGTAATAG
Cd14 GGAAGCCAGAGAACACCATCG GCAGGGCTCCGAATAGAATCC
Lyn GAGAGAAGGCTTCATCCCCAG GTCTTCACAGCCACCTTTGTG
Nek7 GAGCATCCTGTCTCTTGGATG GACTCTTCGAGAATGCATGTG
stella AAAGCTAACCCTAAACCCCGG TGGCTCACTGTCCCGTTCA
vasa CCAAAAGTGACATATATACCC TTGGTTGATCAGTTCTCGAG
Plzf CCACCTTCGCTCACATACAG TGTGGCTCTTGAGTGTGCTC
Taf4b CAGACAGCCAAAGTCAAGCA TGGAGGTCACATGATTCACAC
Oct4 TGAGGCTACAGGACACCTTTC GTGCCAAAGTGGGGACCT
Dazl GCTGATATTTTGCCCAATGAA ATGCTTCGGTCCACAGACTT

Sequences are shown 5′ to 3′.

### Embryonic staging and dissections

The day of appearance of a vaginal plug was taken as E0.5. Plug-positive females were sacrificed by cervical dislocation for collecting blastocysts,E6.5, E12.5, E15.5 and E17.5 embryos. E6.5 and older embryos were genotyped by PCR with primers VP102, VP104, and WF189(Table 1). E12.5, E17.5 and E18.5 embryos were dissected for collection of the embryonic and fetal gonads.

### Immunomagnetic cell-sorting

Total germ cells were prepared as described previously(Vidal et al., 2001). Immunomagnetic isolation of huCD4-positive cells from total testicular cells of Stra8-CD4HAglo mice was performed using the CD4-positive Isolation Kit(Dynal, Oslo, Norway) according to the manufacturer's instructions. For each SSC preparation, 50 transgenic males were used for a final yield of 2×106 cells corresponding to the expected number of 20,000 stem cells per testis (de Rooij and Grootegoed, 1998).

### Purification of spermatids and pachytene spermatocytes

Total germ cells prepared from 20 mice were loaded in the Beckman (Palo Alto, CA, USA) elutriation rotor JE-5.0, at a flow rate of 7 ml/minute and a constant speed of 2000 rpm. Cells were collected in 11 fractions of 400 ml each, obtained by changing the flow rate from 7 to 50 ml/minute at constant speed. Fraction purity was checked by microscopic analysis after Hoechst 33253 staining.

### RNA preparation

Total RNA was extracted from cell fractions using Trizol (Invitrogen, Cergy Pontoise, France) according to the manufacturer's instructions. Ten to 20μg of stem cell RNA was obtained per SSC preparation. Quality of RNA was checked by agarose gel electrophoresis and using an Agilent bioanalyser.

### Histology and immunohistochemistry

Histology and immunohistochemistry analyses were performed according to published protocols (Wagner et al.,2005). Briefly, the samples were fixed overnight at 4°C in paraformaldehyde (3% in PBS) and embedded in paraffin. Routine histological examination was performed after Hematoxylin staining. Sections (3 μm) were incubated (16 hours, 4°C) with primary antibodies against PU.1 (T-21,sc-352, Santa Cruz Biotechnology, Santa Cruz, CA), PCNA (PC10, sc-56, Santa Cruz Biotechnology), each diluted 1:100 in PBS, 0.1% Triton X-100, 3% BSA. Antigen detection was performed with a biotinylated goat anti-rabbit antibody(Vector Laboratories, Burlingame, CA), followed by incubation with peroxidase-coupled streptavidin (Sigma, St Louis, MO). Visualization was achieved with DAB substrate (SK-4100, Vector Laboratories)(Wagner et al., 2006). For PCNA staining, the subsequent antigen detection was performed using the M.O.M. Kit (PK-2200, Vector laboratories). ES cells were stained with the PU.1 antibody as described for U2OS cells(Wagner et al., 2005). Omission of the first antibody served as a negative control; additionally,blocking peptide against PU.1 (sc-352P, Santa Cruz Biotechnology) was used to confirm the specificity of staining.

### Tunel labeling of apoptotic cells

Apoptotic cells were detected by Tunel staining of paraffin sections using the In Situ Cell Death Detection Kit (Roche Molecular Biochemicals) as described previously (Wagner et al.,2005). Three sections from three different animals were analyzed.

### Confocal microscopy

Distribution of GFP fluorescence within the embryo was determined by confocal microscopy with a Leica CLSM microscope equipped with an argon-krypton ion laser emitting light at 488 and 514 nm and producing an excitation wavelength of 515 and 580 nm, and with a UV laser (excitation 352 nm, emission 405 nm). A series of optical sections of approximately 0.95 μm was recorded through the z-axis, using an objective of 43× and a zoom of 1.99.

Fig. 1.

Mouse PU.I (Sfpi1) expression in the huCD4-positive SSC fraction. (A) Germ cell markers in the purified spermatogonial stem cell fraction. RT-PCR assays performed on RNA of huCD4+ cells and of the bulk of differentiated germ cells (huCD4- fraction). Stra8 is expressed in both differentiated and stem spermatogonia(Oulad-Abelghani et al., 1996). Kit is expressed in differentiated spermatogonia, but absent from stem cells(Shinohara et al., 2000). The synaptonemal complex protein 1 gene (Sycp1) is expressed in pachytene spermatocytes (Meuwaissen et al., 1992). Prm1 (protamine 1) is expressed in haploid germ cells (Peschon et al., 1987). Gapdh served as a control. (B)Immunodetection of the PU.1 protein in huCD4+ cells (a) and DAPI staining (b). Note that after immunomagnetic sorting, the cells are still attached to the beads. (C) Quantitative RT-PCR assay of PU.1 RNA in the bulk of differentiated germ cells (huCD4-cells) and in huCD4+ cells. Normalization was performed relative to Gapdh RNA.

Fig. 1.

Mouse PU.I (Sfpi1) expression in the huCD4-positive SSC fraction. (A) Germ cell markers in the purified spermatogonial stem cell fraction. RT-PCR assays performed on RNA of huCD4+ cells and of the bulk of differentiated germ cells (huCD4- fraction). Stra8 is expressed in both differentiated and stem spermatogonia(Oulad-Abelghani et al., 1996). Kit is expressed in differentiated spermatogonia, but absent from stem cells(Shinohara et al., 2000). The synaptonemal complex protein 1 gene (Sycp1) is expressed in pachytene spermatocytes (Meuwaissen et al., 1992). Prm1 (protamine 1) is expressed in haploid germ cells (Peschon et al., 1987). Gapdh served as a control. (B)Immunodetection of the PU.1 protein in huCD4+ cells (a) and DAPI staining (b). Note that after immunomagnetic sorting, the cells are still attached to the beads. (C) Quantitative RT-PCR assay of PU.1 RNA in the bulk of differentiated germ cells (huCD4-cells) and in huCD4+ cells. Normalization was performed relative to Gapdh RNA.

### RT-PCR analysis

DNase I-treated total RNA (1 μg) was reverse-transcribed using the M-MLV reverse transcriptase (Promega, Madison, WI, USA) according to the manufacturer's instructions. PCR amplification was performed with the Ready Mix Red Taq PCR Mix (Sigma) using the oligonucleotide primers shown in Table 1.

### Microarray data analysis

RNA preparations were analyzed by in vitro amplification and hybridization to DNA microarrays containing about 14,000 genes (Mouse Genome 430A 2.0,Affymetrix, Santa Clara, CA, USA). Absolute and comparison analyses of the mouse oligonucleotide arrays were conducted using the statistics-based Affymetrix software MAS-5.0 (GeneChip Software) with the default settings. We used the MAS5 algorithm (Affymetrix) to determine the present' and absent'calls for each probe set. Quantitative expression values were obtained with the robust microarray analysis (RMA) algorithm, which uses a method based on quantile normalization to calculate an expression value for each probe set. The complete microarray data are available in the NCBI GEO repository(http://www.ncbi.nlm.nih.gov/geo/).

### Quantitative real-time PCR

cDNA samples from SSC and total fractions were analyzed by real-time PCR using the qPCR MasterMix Plus for SYBR Green (Eurogentec, Seraing, Belgium)and an ABI Prism 7000 sequence detector according to the manufacturer's protocol (Applied Biosystems, Foster City, CA). Amplification was performed by applying the comparative Ct method, as described in the manufacturer's protocol. Sequences of oligonucleotide primers are shown in Table 1.

### BrdU incorporation

Pregnant female mice received 50 mg/kg of 5-bromo-2′-deoxyuridine(BrdU, Roche Molecular Biochemicals) by intraperitoneal injection and were sacrificed 5 hours later. Paraffin sections were stained by immunolabeling with anti-BrdU antibody (Roche Molecular Biochemicals) as described above.

### Testis transplantation

The donor testes were dissected at E17-19. For transplantation, the recipient testis (6-week-old ICR nu/nu mice, Charles River, Wilmington, MA)was exteriorized through a midline abdominal incision after anaesthetizing the recipient mouse. Using fine forceps, a small cut was made in the tunica albuginea, and the testis graft inserted into the testicular parenchyma. One week to 1 month after transplantation, the recipient testis with the transplant was embedded in paraffin and histological sections were cut at 8-μm intervals from each testis piece and stained with Hematoxylin and Eosin for microscopic examination.

### Preparation and validation of SSC fractions

SSCs were prepared by immunomagnetic sorting with anti-huCD4 antibodies from males of the Stra8-CD4 transgenic family. In these families(Giuili et al., 2002), a neutral surface marker is expressed under control of a truncated version (411 bp) of the promoter of the Stra8 gene. Unlike the wild-type promoter,the truncated version directs transgene expression to a minor population of premeiotic cells, positively identified as SSCs on the basis of expression of marker genes and of their high efficiency of establishment upon transplantation. Among other markers routinely used to ascertain the purity of the fractions (expression of cyclin A2, but not A1, of integrins β1 andα6), the two most characteristic complementary criteria are shown in Fig. 1A, namely expression of the endogenous Stra8 gene, characteristic of both SSCs and differentiated spermatogonia(Oulad-Abdelghani et al.,1996), and absence of Kit transcripts, which are expressed only in differentiated spermatogonia(Shinohara et al., 2000). Contamination by cells engaged in the germinal differentiation process was minimal, as shown by the negative results registered for Sycp1, which is characteristic of the pachytene spermatocytes(Meuwissen et al., 1992) and Prm1, a marker of spermatids(Peschon et al., 1987). Possible contamination by postmeiotic cells was in addition limited by starting the purification process from young males (3-4 weeks), in which the first wave of spermatogenesis would have just passed the second meiotic division (Bellve et al., 1977). Yields were ∼20,000 cells per testis, in agreement with previous estimates(de Rooij and Grootegoed,1998).

### A shortlist of increased in SSCs' transcripts includes PU.1(Sfpi1), a pleiotropic regulator of hematopoiesis

Three independent preparations of 20-40 μg SSC RNA were analyzed by hybridization on DNA microarrays (14,000 genes, Affymetrix Mouse Genome 430A 2.0). In order to identify genes selectively expressed in SSCs, we selected genes that were detected as present' in all three huCD4+ samples and absent' in all three huCD4- samples. We further imposed that the measured detection values exhibit at least a 2-fold difference between huCD4+ and huCD4- samples, with a P-value(Student's t-test) of less than 0.05. These combined criteria led to a list of 84 genes (Table 2). In addition, to estimate the background' of non-specific genes within that list, we performed similar analyses between permuted combinations of samples,in which two samples had been switched between the positive and negative groups. In each case, less then ten genes were found, suggesting that most of the selected genes are indeed differentially expressed between SSCs and non-SSCs. Differential expression of four genes randomly taken in the list(Cd14, Cd97, Nek7, Sfpi1) was also confirmed by quantitative RT-PCR(data not shown). We also noted that a number of genes, although excluded from this most stringent list because of low but significant expression in at least one of the huCD4- samples, showed higher expression levels in the positive fraction. RT-PCR assays of five transcripts in this category(Cd81, Nptn, Fos, Cdc42, Lyn) again showed a differential expression in the two fractions (data not shown).

Fig. 2.

A complex pattern of PU.1 expression in the mouse testis.(A) Immunodetection of the PU.1 protein in the adult (9-month-old)testis, in a fraction of spermatogonia (arrows) and in meiotic spermatocytes(asterisks). (B) A blocking peptide against PU.1 antibody was used to confirm the specifity of staining. (C-E) Expression of PU.1 in meiotic cells during the first synchroneous wave of germinal differentiation(3-week-old testis). (F) RT-PCR assay of PU.1 RNA in fractions enriched by elutriation (≥90%) in pachytene spermatocytes (lane 1) and elongated and round spermatids (lanes 2 and 3, respectively). (G-I)Expression in single (G), paired (H) and aligned (I) cells (arrows) in the peripheral compartment of the seminiferous tubule (Asingle,Apaired and Aaligned spermatogonia). Scale bars: 50μm in A-E; 25 μm in G-I.

Fig. 2.

A complex pattern of PU.1 expression in the mouse testis.(A) Immunodetection of the PU.1 protein in the adult (9-month-old)testis, in a fraction of spermatogonia (arrows) and in meiotic spermatocytes(asterisks). (B) A blocking peptide against PU.1 antibody was used to confirm the specifity of staining. (C-E) Expression of PU.1 in meiotic cells during the first synchroneous wave of germinal differentiation(3-week-old testis). (F) RT-PCR assay of PU.1 RNA in fractions enriched by elutriation (≥90%) in pachytene spermatocytes (lane 1) and elongated and round spermatids (lanes 2 and 3, respectively). (G-I)Expression in single (G), paired (H) and aligned (I) cells (arrows) in the peripheral compartment of the seminiferous tubule (Asingle,Apaired and Aaligned spermatogonia). Scale bars: 50μm in A-E; 25 μm in G-I.

Fig. 3.

Immunodetection of PU.1 in the fetal and neonate mouse testis.Immunodetection was performed at E12.5 (A,B), E15.5(C,D) and at postnatal day 5 (E,F). Scale bars: 50μm.

Fig. 3.

Immunodetection of PU.1 in the fetal and neonate mouse testis.Immunodetection was performed at E12.5 (A,B), E15.5(C,D) and at postnatal day 5 (E,F). Scale bars: 50μm.

Fig. 4.

Abnormal testicular development of the PU.1-negative mouse mutant. (Aa-g,Ba-g) Testis sections of PU.1+/+ (a,c,e) and PU.1G/G (b,d,f)littermates at E15.5 (A) and E17.5 (B). (Ag,Bg) The average number of gonocytes per transverse tubule section, for both developmental times.(C) RT-PCR assay for vasa and stella expression performed on RNA prepared from E18.5 PU.1G/G, PU.1G/+ and PU.1+/+ fetal testis. Scale bars: 50 μm.

Fig. 4.

Abnormal testicular development of the PU.1-negative mouse mutant. (Aa-g,Ba-g) Testis sections of PU.1+/+ (a,c,e) and PU.1G/G (b,d,f)littermates at E15.5 (A) and E17.5 (B). (Ag,Bg) The average number of gonocytes per transverse tubule section, for both developmental times.(C) RT-PCR assay for vasa and stella expression performed on RNA prepared from E18.5 PU.1G/G, PU.1G/+ and PU.1+/+ fetal testis. Scale bars: 50 μm.

Table 2.

Transcripts preferentially expressed in huCD4-positive cells

Probe set number*GeneSymbolFold change (log2)
1422903_at lymphocyte antigen 86 Ly86 4.08
1419627_s_at C-type lectin domain family 4, member n Clec4n 3.82
1448148_at granulin Grn 3.80
1448617_at CD53 antigen Cd53 3.74
1450241_a_at ecotropic viral integration site 2a Evi2a 3.61
1454268_a_at cytochrome b-245, alpha polypeptide Cyba 3.54
1451941_a_at Fc receptor, IgG, low affinity llb Fcgr2b 3.52
1448891_at macrophage scavenger receptor 2 Msr2 3.38
1417963_at phospholipid transfer protein Pltp 3.30
1431724_a_at purinergic receptor P2Y, G-protein coupled 12 P2ry12 3.26
1449556_at histocompatibility 2, T region locus 23 H2-T23 3.25
1425951_a_at C-type lectin domain family 4, member n Clec4n 3.20
1426454_at Rho, GDP dissociation inhibitor (GDI) beta Arhgdib 3.18
1450678_at integrin beta 2 Itgb2 3.01
1425519_a_at CD74 antigen Cd74 2.97
1427076_at macrophage expressed gene 1 Mpeg1 2.94
1449918_at CD209g antigen Cd209g 2.86
1419482_at complement component 3a receptor 1 C3ar1 2.84
1416326_at cysteine-rich protein 1 (intestinal) Crip1 2.79
1425214_at pyrimidinergic receptor P2Y, G-protein coupled, 6 P2ry6 2.72
1423805_at disabled homolog 2 (DrosophilaDab2 2.70
1449971_a_at CD209f antigen Cd209f 2.54
1452544_x_at histocompatibility 2, D region locus 1 H2-D1 2.47
1435476_a_at Fc receptor, IgG, low affinity llb Fcgr2b 2.46
1451386_at biliverdin reductase B [flavin reductase (NADPH)] Blvrb 2.43
1419043_a_at interferon inducible GTPase 1 ligp1 2.42
1427329_a_at immunoglobulin heavy chain 6 (heavy chain of IgM) lgh-6 2.41
1423266_at RIKEN cDNA 2810405K02 gene 2810405K02Rik 2.37
1450919_at membrane protein, palmitoylated Mpp1 2.31
1416246_a_at coronin, actin binding protein 1A Coro1a 2.29
1427301_at CD48 antigen Cd48 2.28
1452231_x_at interferon activated gene 203 lfi203 2.19
1417268_at CD14 antigen Cd14 2.18
1448160_at lymphocyte cytosolic protein 1 Lcp1 2.13
1424542_at S100 calcium binding protein A4 S100a4 2.07
1419027_s_at glycolipid transfer protein Gltp 2.06
1417869_s_at cathepsin Z Ctsz 2.06
1416816_at NIMA (never in mitosis gene a)-related expressed kinase 7 Nek7 2.05
1448944_at neuropilin 1 Nrp1 1.96
1419144_at CD 163 antigen Cd163 1.95
1448276_at tetraspanin 4 Tspan4 1.92
1435375_at cDNA sequence BC052328 BC052328 1.88
1426918_at integrin beta 1 (fibronectin receptor beta) ltgb1 1.88
1451683_x_at similar to H-2 class I histocompatibility antigen, L-D alpha chain precursor LOC547343 1.86
1417491_at cathepsin B Ctsb 1.84
1434036_at metastasis suppressor 1 Mtss1 1.81
1448748_at pleckstrin Plek 1.77
1419282_at chemokine (C-C motif) ligand 12 Ccl12 1.77
1416985_at signal-regulatory protein alpha Sirpa 1.77
1455269_a_at coronin, actin binding protein 1A Coro1a 1.77
1423166_at CD36 antigen Cd36 1.72
1423570_at ATP-binding cassette, sub-family G (WHITE), member 1 Abcg1 1.70
1418641_at lymphocyte cytosolic protein 2 Lcp2 1.65
1419120_at lymphoblastomic leukemia Lyl1 1.63
1423653_at ATPase, Na+/K+ transporting, alpha 1 polypeptide Atp1a1 1.61
1417162_at transmembrane BAX inhibitor motif containing 1 Tmbim1 1.52
1450040_at tissue inhibitor of metalloproteinase 2 Timp2 1.51
1448239_at heme oxygenase (decycling) 1 Hmox1 1.50
1455393_at 1.49
1417676_a_at protein tyrosine phosphatase, receptor type, O Ptpro 1.46
1417561_at apolipoprotein C-l Apoc1 1.44
1420464_s_at paired-lg-like receptor A1 Pira1 1.44
1418747_at SFFV proviral integration 1 Sfpi1 1.40
1420911_a_at milk fat globule-EGF factor 8 protein Mfge8 1.38
1428340_s_at ATPase type 13A2 Atp13a2 1.36
1450107_a_at renin binding protein Renbp 1.34
1452169_a_at diacylglycerol kinase zeta Dgkz 1.25
1416897_at poly (ADP-ribose) polymerase family, member 9 Parp9 1.14
1428369_s_at Rho GTPase activating protein 21 Arhgap21 1.14
1448797_at ELK3, member of ETS oncogene family Elk3 1.14
1428369_s_at Rho GTPase activating protein 21 Arhgap21 1.14
1448797_at ELK3, member of ETS oncogene family Elk3 1.14
1415892_at sphingosine phosphate lyase 1 Sgp/1 1.14
1423757_x_at insulin-like growth factor binding protein 4 lgfbp4 1.14
1424778_at DNA segment, Chr 10, University of California at Los Angeles 1 Reep3 1.13
1424394_at selenoprotein M Selm 1.12
1451041_at Rho-associated coiled-coil containing protein kinase 2 Rock2 1.11
1423153_x_at complement component factor h Cfh 1.11
1450026_a_at UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2 B3gnt2 1.09
1426663_s_at solute carrier family 45, member 3 Slc45a3 1.08
1425609_at neutrophil cytosolic factor 1 Ncf1 1.07
1426376_at receptor accessory protein 5 Reep5 1.07
1450105_at 1.07
1448957_at recombining binding protein suppressor of hairless (DrosophilaRbpsuh 1.05
1418394_a_at CD97 antigen Cd97 1.04
1424769_s_at caldesmon 1 Cald1 1.01
Probe set number*GeneSymbolFold change (log2)
1422903_at lymphocyte antigen 86 Ly86 4.08
1419627_s_at C-type lectin domain family 4, member n Clec4n 3.82
1448148_at granulin Grn 3.80
1448617_at CD53 antigen Cd53 3.74
1450241_a_at ecotropic viral integration site 2a Evi2a 3.61
1454268_a_at cytochrome b-245, alpha polypeptide Cyba 3.54
1451941_a_at Fc receptor, IgG, low affinity llb Fcgr2b 3.52
1448891_at macrophage scavenger receptor 2 Msr2 3.38
1417963_at phospholipid transfer protein Pltp 3.30
1431724_a_at purinergic receptor P2Y, G-protein coupled 12 P2ry12 3.26
1449556_at histocompatibility 2, T region locus 23 H2-T23 3.25
1425951_a_at C-type lectin domain family 4, member n Clec4n 3.20
1426454_at Rho, GDP dissociation inhibitor (GDI) beta Arhgdib 3.18
1450678_at integrin beta 2 Itgb2 3.01
1425519_a_at CD74 antigen Cd74 2.97
1427076_at macrophage expressed gene 1 Mpeg1 2.94
1449918_at CD209g antigen Cd209g 2.86
1419482_at complement component 3a receptor 1 C3ar1 2.84
1416326_at cysteine-rich protein 1 (intestinal) Crip1 2.79
1425214_at pyrimidinergic receptor P2Y, G-protein coupled, 6 P2ry6 2.72
1423805_at disabled homolog 2 (DrosophilaDab2 2.70
1449971_a_at CD209f antigen Cd209f 2.54
1452544_x_at histocompatibility 2, D region locus 1 H2-D1 2.47
1435476_a_at Fc receptor, IgG, low affinity llb Fcgr2b 2.46
1451386_at biliverdin reductase B [flavin reductase (NADPH)] Blvrb 2.43
1419043_a_at interferon inducible GTPase 1 ligp1 2.42
1427329_a_at immunoglobulin heavy chain 6 (heavy chain of IgM) lgh-6 2.41
1423266_at RIKEN cDNA 2810405K02 gene 2810405K02Rik 2.37
1450919_at membrane protein, palmitoylated Mpp1 2.31
1416246_a_at coronin, actin binding protein 1A Coro1a 2.29
1427301_at CD48 antigen Cd48 2.28
1452231_x_at interferon activated gene 203 lfi203 2.19
1417268_at CD14 antigen Cd14 2.18
1448160_at lymphocyte cytosolic protein 1 Lcp1 2.13
1424542_at S100 calcium binding protein A4 S100a4 2.07
1419027_s_at glycolipid transfer protein Gltp 2.06
1417869_s_at cathepsin Z Ctsz 2.06
1416816_at NIMA (never in mitosis gene a)-related expressed kinase 7 Nek7 2.05
1448944_at neuropilin 1 Nrp1 1.96
1419144_at CD 163 antigen Cd163 1.95
1448276_at tetraspanin 4 Tspan4 1.92
1435375_at cDNA sequence BC052328 BC052328 1.88
1426918_at integrin beta 1 (fibronectin receptor beta) ltgb1 1.88
1451683_x_at similar to H-2 class I histocompatibility antigen, L-D alpha chain precursor LOC547343 1.86
1417491_at cathepsin B Ctsb 1.84
1434036_at metastasis suppressor 1 Mtss1 1.81
1448748_at pleckstrin Plek 1.77
1419282_at chemokine (C-C motif) ligand 12 Ccl12 1.77
1416985_at signal-regulatory protein alpha Sirpa 1.77
1455269_a_at coronin, actin binding protein 1A Coro1a 1.77
1423166_at CD36 antigen Cd36 1.72
1423570_at ATP-binding cassette, sub-family G (WHITE), member 1 Abcg1 1.70
1418641_at lymphocyte cytosolic protein 2 Lcp2 1.65
1419120_at lymphoblastomic leukemia Lyl1 1.63
1423653_at ATPase, Na+/K+ transporting, alpha 1 polypeptide Atp1a1 1.61
1417162_at transmembrane BAX inhibitor motif containing 1 Tmbim1 1.52
1450040_at tissue inhibitor of metalloproteinase 2 Timp2 1.51
1448239_at heme oxygenase (decycling) 1 Hmox1 1.50
1455393_at 1.49
1417676_a_at protein tyrosine phosphatase, receptor type, O Ptpro 1.46
1417561_at apolipoprotein C-l Apoc1 1.44
1420464_s_at paired-lg-like receptor A1 Pira1 1.44
1418747_at SFFV proviral integration 1 Sfpi1 1.40
1420911_a_at milk fat globule-EGF factor 8 protein Mfge8 1.38
1428340_s_at ATPase type 13A2 Atp13a2 1.36
1450107_a_at renin binding protein Renbp 1.34
1452169_a_at diacylglycerol kinase zeta Dgkz 1.25
1416897_at poly (ADP-ribose) polymerase family, member 9 Parp9 1.14
1428369_s_at Rho GTPase activating protein 21 Arhgap21 1.14
1448797_at ELK3, member of ETS oncogene family Elk3 1.14
1428369_s_at Rho GTPase activating protein 21 Arhgap21 1.14
1448797_at ELK3, member of ETS oncogene family Elk3 1.14
1415892_at sphingosine phosphate lyase 1 Sgp/1 1.14
1423757_x_at insulin-like growth factor binding protein 4 lgfbp4 1.14
1424778_at DNA segment, Chr 10, University of California at Los Angeles 1 Reep3 1.13
1424394_at selenoprotein M Selm 1.12
1451041_at Rho-associated coiled-coil containing protein kinase 2 Rock2 1.11
1423153_x_at complement component factor h Cfh 1.11
1450026_a_at UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2 B3gnt2 1.09
1426663_s_at solute carrier family 45, member 3 Slc45a3 1.08
1425609_at neutrophil cytosolic factor 1 Ncf1 1.07
1426376_at receptor accessory protein 5 Reep5 1.07
1450105_at 1.07
1448957_at recombining binding protein suppressor of hairless (DrosophilaRbpsuh 1.05
1418394_a_at CD97 antigen Cd97 1.04
1424769_s_at caldesmon 1 Cald1 1.01
*

Probe set number relative to the Mouse Genome 430A 2.0 (Affymetrix).

Official gene name and symbol according to NCBI.

Fold change (log2) between huCD4-positive and -negative samples.

Fig. 5.

Expression of early germinal markers in the fetal PU.1G/G mouse testis. (A-F) Quantitative RT-PCR determination at E18.5 relative to β-actin RNA. Oligonucleotide primers are listed in Table 1.

Fig. 5.

Expression of early germinal markers in the fetal PU.1G/G mouse testis. (A-F) Quantitative RT-PCR determination at E18.5 relative to β-actin RNA. Oligonucleotide primers are listed in Table 1.

Several genes encoding transcription factors were present among the genes in the most stringent shortlist: Lyl1 encoding a bHLH factor(Capron et al., 2006), Elk3 encoding an Ets-domain factor(Chung et al., 2006) and Rbpsuh (Rbpj) encoding the transcriptional mediator of Notch signaling (Vasyutina et al.,2007).

Although all of these genes might play important regulatory roles in controlling SSC gene expression, we were especially intrigued by the presence of PU.1 (Sfpi1). This finding warranted further analysis because the published literature (reviewed by Lloberas et al., 1999; Metcalf et al., 2006)consistently refers to PU.1 as a transcription regulator that acts exclusively in the progenitors of blood cells. Both PU.1 RNA and the protein were unequivocally detected in huCD4+ cells by quantitative RT-PCR and by immunolabeling (Fig. 1B,C).

### PU.1 expression in the adult testis

Immunodetection of PU.1 protein in adult testis sections revealed a complex pattern. As expected from analysis of the huCD4+ fraction, the protein was detected in a small number of spermatogonia in the most peripheral region of the tubule (Fig. 2A). Among these cells, the paired (Apaired) and aligned(Aaligned) cells characteristic of the early spermatogonial stages(de Rooij and Grootegoed,1998) were clearly identified(Fig. 2G-I). Whereas the bulk of differentiated spermatogonia were negative, expression of the protein was also evidenced in meiotic spermatocytes(Fig. 2A, asterisk). This finding was somewhat unexpected, a contamination by pachytene spermatocytes of the huCD4+ fraction being made unlikely by the absence of the characteristic meiotic transcripts Sycp1(Fig. 1A) and Ccna2(cyclin A2, not shown) (see Giuili et al.,2002). Meiotic expression was nevertheless confirmed by in situ analysis in the testis of 3-week-old males, in which the first synchronous wave of spermatogenesis leads to the accumulation of meiotic cells, which were uniformly and strongly labeled by anti-PU.1 immunostaining(Fig. 2C-E). This was further confirmed by PCR assays performed on elutriation-purified spermatocytes(Fig. 2F). The quantitatively minor representation of pachytene spermatocytes in the total huCD4-fraction accounts for the initial detection of higher levels of PU.1transcripts in huCD4+ cells.

### PU.1 activity during development is required in germinal differentiation

In the embryonic testis, PU.1 was detected in the germinal compartment, at least from E12.5 onward (Fig. 3). Further studies took advantage of a mouse mutant, PU.1G, generated by insertion of a GFP cassette into the first exon of the gene to provide both a reporter for promoter activity and a null mutation (Back et al.,2004). PU.1G/G homozygotes die shortly after birth and heterozygotes are healthy and fertile. Embryos of crosses between heterozygotes were individually genotyped and testis development was analyzed in the PU.1G/G progeny, with their G/+ and +/+littermates used as controls. Histological analysis of homozygotes at E15.5 and E17.5 seemed at first to indicate that the testis was normally constituted, with a myoid cell layer surrounding internal cells in a structure similar to the testis cords of the wild type(Fig. 4A,B). The number of gonocytes per section was, however, smaller than in the controls. Among a series of genes characteristic of prenatal germinal differentiation, RT-PCR analysis detected transcripts of Oct4(Yoshimizu et al., 1999), Dazl (Lin and Page,2005), Taf4b(Falender et al., 2005) and vasa (Tanaka et al., 2000),but expression of Plzf (Zbtb16)(Buaas et al., 2004; Costoya et al., 2004) and stella (Saitou et al., 2002)was strongly reduced or absent in the null mutant(Fig. 4C and Fig. 5).

The defect in spermatogenesis of the PU.1G/G mutant was related to a decrease in cell proliferation, as evidenced by decreased BrdU incorporation during development (Fig. 6). The number of germ cells incorporating BrdU was significantly decreased at E12.5 and E13.5. By contrast, BrdU incorporation in the somatic component of the testis cords (essentially Sertoli cells) was decreased to a lesser extent and only at later times, an observation that will be addressed in future studies. At postnatal stages, a functional defect of PU.1G/G testes was evidenced by testis graft experiments,an approach made necessary by the early death of the mutant homozygotes. Testes dissected shortly before birth (E18.5) were grafted under the tunica albuginea of recipient nude mice. Spermatogenesis is known to proceed normally in such grafts up to the production of functional sperm(Honaramooz et al., 2002). Grafts of the PU.1G/+ testes showed, after 1 week, a strong proliferative activity as evidenced by PCNA immunolabeling(Fig. 7Ae,g), leading, after 1 month, to the development of a structurally normal seminiferous epithelium(Fig. 7Ba,c). By contrast,grafts of their PU.1G/G littermates showed, 1 week after transplantation, only a limited proliferative activity of Sertoli cells in the periphery of the tubules (Fig. 7f,h), which were essentially empty after 2 weeks (not shown) and 4 weeks (Fig. 7Bb,d). Thus, not only was the number of gonocytes reduced owing to a decreased proliferation rate, but the surviving germ cells were unable to enter a normal differentiation pathway.

Fig. 6.

Decreased proliferation and absence of apoptosis of male germ cells in the PU.1-negative mouse mutant. (Aa-i) BrdU incorporation was monitored by immunolabeling after 5-hour pulses at E12.5, E13.5 and E15.5 in PU.1G/G homozygotes (b,e,h) and their PU.1+/+ littermates (a,d,g). (c,f,i) Quantitation by counts of positive cells in whole-gonad sections (including germ cells and somatic cells). The faint staining of the mutant cells counted as positive at E12.5 may reflect either lower rates of replication or repair activity. Note that the label in the mutant testis at E13.5 and E15.5 is restricted to the peripheral somatic cells. (Ba-f) Tunel assay for apoptotic cells was performed on E15.5 (a,b) and E17.5 (d,e) embryos. (c,f) Quantitation of Tunel-positive cells. Scale bars: 50 μm.

Fig. 6.

Decreased proliferation and absence of apoptosis of male germ cells in the PU.1-negative mouse mutant. (Aa-i) BrdU incorporation was monitored by immunolabeling after 5-hour pulses at E12.5, E13.5 and E15.5 in PU.1G/G homozygotes (b,e,h) and their PU.1+/+ littermates (a,d,g). (c,f,i) Quantitation by counts of positive cells in whole-gonad sections (including germ cells and somatic cells). The faint staining of the mutant cells counted as positive at E12.5 may reflect either lower rates of replication or repair activity. Note that the label in the mutant testis at E13.5 and E15.5 is restricted to the peripheral somatic cells. (Ba-f) Tunel assay for apoptotic cells was performed on E15.5 (a,b) and E17.5 (d,e) embryos. (c,f) Quantitation of Tunel-positive cells. Scale bars: 50 μm.

### PU.1 expression in early embryonic stages and embryonic stem cells

Although not absolutely required for spermatogenesis(Bortvin et al., 2004), stella is the earliest marker of embryonic differentiation; it is already expressed in the blastocyst and later restricted to the germinal lineage(Saitou et al., 2002; Sato et al., 2002) (our unpublished results). The correlation between PU.1 and stella expression at the late fetal stage led us to check whether, as is stella itself, PU.1 is a marker of early embryonic development. Confocal microscopy detected GFP fluorescence in PU.1G/+heterozygotes as early as the blastocyst stage (E3.5), at which the inner cell mass showed heterogeneous but strong labeling(Fig. 8A). ES cells were accordingly checked and found positive by immunolabeling with anti-PU.1 antibodies (Fig. 8B). In the E6.5 postimplantation embryo (Fig. 8C), GFP fluorescence indicated extensive expression in the epiblast and extraembryonic ectoderm, with a marked concentration in the general area of the node, which was maintained during the following days in a variety of structures. Although both genes are expressed at the same early stage, we could exclude the possibility that the PU.1 protein directly controls stella transcription, as transcripts of the latter were clearly detected in the early PU.1G/G embryo (E10.5, data not shown). The pattern of PU.1 expression is in fact distinct and more extensive than that of stella, Blimp1 and fragilis, currently considered as the relevant markers of germ line specification and similarly expressed at very early stages (Saitou et al., 2002).

Fig. 7.

Arrest of spermatogenesis in the PU.1 mutant gonad.(Aa-h) Wild-type E18.5 testes (a,c,e,g) and testes of their PU.1G/G littermates (b,d,f,h) were dissected and grafted under the tunica albuginea of recipient nude mice. Histological analysis and PCNA immunostaining were performed 1 week after transplantation. The contorted shape of the peripheral labeled nuclei in h is a known characteristic of Sertoli cells. (Ba-d) The same analysis 4 weeks after grafting. At low magnification, the limit of the graft (upper left) and the host tissue (lower right) can be seen in a and b, corresponding in b to that between empty tubules and normal spermatogenesis. Scale bars: 200 μm in Aa,Ab,Ba,Bb; 50μm in all other panels.

Fig. 7.

Arrest of spermatogenesis in the PU.1 mutant gonad.(Aa-h) Wild-type E18.5 testes (a,c,e,g) and testes of their PU.1G/G littermates (b,d,f,h) were dissected and grafted under the tunica albuginea of recipient nude mice. Histological analysis and PCNA immunostaining were performed 1 week after transplantation. The contorted shape of the peripheral labeled nuclei in h is a known characteristic of Sertoli cells. (Ba-d) The same analysis 4 weeks after grafting. At low magnification, the limit of the graft (upper left) and the host tissue (lower right) can be seen in a and b, corresponding in b to that between empty tubules and normal spermatogenesis. Scale bars: 200 μm in Aa,Ab,Ba,Bb; 50μm in all other panels.

Previous work underlined the pleiotropic roles of the PU.1 protein, which exerts distinct functions in the progenitors of the various hematopoietic lineages (reviewed by Lloberas et al.,1999; Metcalf et al.,2006). The picture must now be expanded to include other classes of stem cells, at least the SSCs of the adult mammalian testis and embryonic PGCs. The list of progenitors under PU.1 control might not yet be complete,considering the widespread expression of the gene during embryogenesis. Considering the lineages for which sufficient data exist, namely the lymphopoietic and erythropoietic progenitors(Back et al., 2004; Iwasaki et al., 2005) and now the germinal lineage, a common picture seems to be emerging, in which the gene would act in the maintenance of cells in the undifferentiated state.

In the germinal differentiation process, our initial finding was a regulated expression of PU.1 RNA in the adult testis, first in SSCs and early spermatogonia, and later in meiotic spermatocytes. The availability of the null mutant PU.1G was initially thought to provide the right tool to unravel its role in these cell types. It turned out,however, that expression of the locus starts at a much earlier stage of germinal differentiation - in fact, at the time of the first specification of germ cells and even before. Furthermore, although the structure of the fetal testis appeared at least grossly normal, it became clear that proliferation of the germ cells was strongly affected, resulting in a much reduced number of gonocytes at the end of fetal development. The remaining cells were in fact unable to enter the normal differentiation pathway, the lack of recognizable germinal progression in the grafted testes defining an absolute requirement for PU.1 expression in the fetal differentiation of germ cells.

Fig. 8.

PU.1 promoter expression in the early mouse embryo and ES cells. (A) GFP fluorescence in PU.1G/+heterozygote E3.5 blastocyst determined by confocal microscopy. (B)Immunodetection of the PU.1 protein in ES cells. (C) Confocal microscopy detection of GFP fluorescence in two optical sections of the same E6.5 PU.1G/+ postimplantation embryo (left, tangential upper section; right, middle section). Arrow shows the area of the node that displays a marked concentration of GFP-positive cells in an as yet undefined structure. Images shown in A and C are representative of those from a series of 20-30 embryos from three litters. Scale bars: 20 μm in A,B; 80 μm in C.

Fig. 8.

PU.1 promoter expression in the early mouse embryo and ES cells. (A) GFP fluorescence in PU.1G/+heterozygote E3.5 blastocyst determined by confocal microscopy. (B)Immunodetection of the PU.1 protein in ES cells. (C) Confocal microscopy detection of GFP fluorescence in two optical sections of the same E6.5 PU.1G/+ postimplantation embryo (left, tangential upper section; right, middle section). Arrow shows the area of the node that displays a marked concentration of GFP-positive cells in an as yet undefined structure. Images shown in A and C are representative of those from a series of 20-30 embryos from three litters. Scale bars: 20 μm in A,B; 80 μm in C.

A relationship between PU.1 and stella expression had been considered because of the concomitant expression of the genes at very early developmental stages and by the absence of stella RNA in the fetal testis of the PU.1-null mutants. A direct transcriptional regulation of stella by the PU.1 protein was, however, excluded by the observation that the gene is expressed in the early embryo (E7.5) of the PU.1-negative mutant. It must also be remembered that abrogation of stella expression does not affect germinal differentiation (Bortvin et al.,2004), and we also noted that the widespread expression of PU.1 in the postimplantation embryo does not correspond to that of stella, which is rapidly restricted to the germinal lineage.

The finding that PU.1 is required for the embryonic differentiation of germ cells leaves open the question of its role in the adult testis. An analysis would require a conditional mutation in which gene expression would be arrested exclusively after birth. An intriguing and complex pattern, with expression in SSCs and possibly early spermatogonia, extinguished in differentiated spermatogonia and starting again in meiotic cells, might actually suggest distinct activities in these different cell types. Altogether, a relatively extended domain of expression, starting at the earliest stages of development and required in at least two independent lineages, suggests a common function in the maintenance of embryonic and adult stem cells and in the control of their differentiation. Given the multiplicity of protein variants encoded by the PU.1 gene(Lloberas et al., 1999), it remains to be determined whether they exert distinct functions in different lineages.

We thank Yan Fantei-Caujolles and Sandra Kanani for expert handling of databases; Jean Claude Hammou for pathological expertise; Christelle Thibault for the generation of microarray data; Cedric Mathews for confocal microscopy analysis; and K. D. Wagner for critical reading of the manuscript. V.O. was supported by scholarships from Région PACA-Inserm and from Association pour la Recherche sur le Cancer, France. N.W. is a fellow of Fondation de France and was supported financially by A. Schedl. This work was made possible by a grant of Ligue Nationale Contre le Cancer as Equipe Labellisée and by grants from Ministère de la Recherche to M.R. and from Inserm (ACI FNS2001) to F.C.

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