spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 3 October 2007
doi: 10.1242/dev.003467

Development 134, 3815-3825 (2007)
Published by The Company of Biologists 2007
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Olive, V.
Right arrow Articles by Rassoulzadegan, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Olive, V.
Right arrow Articles by Rassoulzadegan, M.

PU.1 (Sfpi1), a pleiotropic regulator expressed from the first embryonic stages with a crucial function in germinal progenitors

Virginie Olive1,2,*, Nicole Wagner1,2,*, Susan Chan3, Philippe Kastner3, Christine Vannetti1,2, François Cuzin1,2 and Minoo Rassoulzadegan1,2,{dagger}

1 Inserm U636, F-06108, Nice, France.
2 Université de Nice-Sophia Antipolis, Laboratoire de Génétique du Développement Normal et Pathologique, F-06108 Nice, France.
3 Institut de Biologie Moléculaire et Cellulaire CNRS-INSERM-ULP, Illkirch, F-67404, France.


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		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., 2000Go). 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., 1987Go). 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.

 

Figure 2
View larger version (107K):
[in this window]
[in a new window]

  		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: 50 µm in A-E; 25 µm in G-I.

 

Figure 3
View larger version (157K):
[in this window]
[in a new window]

  		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: 50 µm.

 

Figure 4
View larger version (85K):
[in this window]
[in a new window]

  		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.

 

Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		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 ß-actin RNA. Oligonucleotide primers are listed in Table 1.

 

Figure 6
View larger version (77K):
[in this window]
[in a new window]

  		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.

 

Figure 7
View larger version (69K):
[in this window]
[in a new window]

  		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; 50 µm in all other panels.

 

Figure 8
View larger version (137K):
[in this window]
[in a new window]

  		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.

 




© The Company of Biologists Ltd 2007