Disruption of genital ridge development causes aberrant primordial germ cell proliferation but does not affect their directional migration
© Chen et al; licensee BioMed Central Ltd. 2013
Received: 21 January 2013
Accepted: 5 March 2013
Published: 5 March 2013
The directional migration and the following development of primordial germ cells (PGCs) during gonad formation are key steps for germline development. It has been proposed that the interaction between germ cells and genital ridge (GR) somatic cells plays essential roles in this process. However, the in vivo functional requirements of GR somatic cells in germ cell development are largely unknown.
Wt1 mutation (Wt1R394W/R394W) results in GR agenesis through mitotic arrest of coelomic epitheliums. In this study, we employed the GR-deficient mouse model, Wt1R394W/R394W, to investigate the roles of GR somatic cells in PGC migration and proliferation. We found that the number of PGCs was dramatically reduced in GR-deficient embryos at embryonic day (E) 11.5 and E12.5 due to decreased proliferation of PGCs, involving low levels of BMP signaling. In contrast, the germ cells in Wt1R394W/R394W embryos were still mitotically active at E13.5, while all the germ cells in control embryos underwent mitotic arrest at this stage. Strikingly, the directional migration of PGCs was not affected by the absence of GR somatic cells. Most of the PGCs reached the mesenchyme under the coelomic epithelium at E10.5 and no ectopic PGCs were noted in GR-deficient embryos. However, the precise positioning of PGCs was disrupted.
Our work provides in vivo evidence that the proliferation of germ cells is precisely regulated by GR somatic cells during different stages of gonad development. GR somatic cells are probably dispensable for the directional migration of PGCs, but they are required for precise positioning of PGCs at the final step of migration.
KeywordsPrimordial germ cells Genital ridge Migration, Proliferation
Primordial germ cells (PGCs) are the precursors of spermatozoa and oocytes, which are derived from a small number of epiblast cells under induction of bone morphogenetic protein (BMP) signaling and other unidentified signals from the extraembryonic ectoderm (ExE) and visceral endoderm (VE) at embryonic day (E) 6.5 in mice [1, 2]. Following specification, PGCs must become motile and actively migrate across the embryo to reach the developing genital ridge (GR) and form the functional gonads in combination with surrounding somatic cells . In mice, the PGCs move from the primitive streak to the endoderm at E7.5 . Then, they migrate through the hindgut and mesentery and arrive at the GR at approximately E10.5 .
Previous studies have suggested that the directional migration of PGCs towards the GR is regulated by a combination of attractive and repulsive signals [6–8]. Stromal cell-derived factor 1 (Sdf1) is the most promising attractant signaling candidate, which is primarily expressed in the GR and surrounding mesenchyme; its receptor, Cxcr4, is expressed in PGCs [9–11]. The importance of this chemokine signaling for PGCs migration and colonization has been demonstrated by studying gene knockout mouse models [9, 11]. c-kit is another gene that has been implicated in guiding mouse PGC migration. c-kit is expressed in PGCs and its ligand, Steel factor, is expressed by somatic cells along the migratory route . Other than attractive signals, the adhesion molecule E-cadherin [13, 14] and extracellular matrix molecule Integrin β1  have been reported to be involved in the regulation of PGC migration and colonization in GR. However, the precise function of GR somatic cells in PGCs migration remains to be elucidated.
Once the PGCs reach the GR, they lose their motility and proliferate rapidly [16, 17]. BMP signaling has been shown to control formation of the PGC niche and proliferation of PGCs within the early GR [18, 19]. After sex determination, the germ cells in both male and female embryos cease proliferation. Male germ cells arrest at the G0 phase of mitosis, while female germ cells initiate meiosis and arrest at the diplotene stage of prophase I [20, 21]. Whether the proliferation and differentiation of PGCs is a cell autonomous process [22–26] or is under the control of the surrounding gonadal somatic cells  remains an open question.
The Wilms' tumor (WT) suppressor gene, Wt1, encodes a nuclear zinc finger transcription factor that was originally identified as a tumor suppressor gene in patients with WT [28–30]. Wt1 has been implicated in the regulation of target genes related to proliferation and cell cycle progression [31, 32]. Wt1 is expressed in the urogenital ridge coelomic epithelium and the underlying mesenchymal cells during embryo development . It has been reported that Wt1 is essential for GR development and deletion of Wt1 results in gonadal agenesis due to the failure of GR development . The alkaline phosphatase-positive PGCs are observed in the mesenchyme close to coelomic epithelium in Wt1-/- embryo at E12, suggesting that aberrant GR development probably does not interfere with the germ cell migration . However, whether the germ cell migrating process is normal and all the germ cells reach the aberrant GR; or the proliferation of germ cells has been affected in Wt1-/- embryo remains unclear. Wt1R394W mice carry the most common Denys-Drash syndrome (DDS) missense mutation . Wt1 +/R394W mice display severe renal failure, and Wt1R394W/R394W mice are embryonically lethal . Agenesis of gonads and kidneys is also noted in Wt1R394W/R394W mice due to aberrant GR development which is similar to Wt1-null mice.
In this study we found that the migrating process of PGCs was normal and no ectopic PGCs were observed in Wt1R394W/R394W. However, the number of PGCs in Wt1R394W/R394W embryos was dramatically reduced compared to control embryos due to the mitotic arrest. In contrast, abnormal proliferation of PGCs was also observed in Wt1R394W/R394W embryos at E13.5. These results suggest that the proliferation of germ cells during the early stage of gonad development is precisely regulated by GR somatic cells, and the GR somatic cells are probably dispensable for directional migration of PGCs.
Wt1 mutation (Wt1R394W/R394W) results in GR agenesis through mitotic arrest of coelomic epitheliums
The GR arises as a thickening of the epithelium along the coelomic surface of the mesonephros [33, 35]. A previous study suggests that Wt1 plays a critical role in GR development, as deletion of Wt1 results in aberrant GR development and absence of kidneys and gonads in mice . The authors speculate that loss of Wt1 probably leads to apoptosis of coelomic epithelial cells, which in turn causes GR agenesis in Wt1-/- mice . However, the exact function of Wt1 in GR development is unclear.
Wt1 has been implicated in the regulation of target genes related to cell cycle progression [31, 32] and Cyclin E1 has been demonstrated to play a crucial role in the cell cycle by binding cycle-dependent kinase 2 (CDK2), which phosphorylates Rb, leading to transition from G1 into S phase . To identify the underlying mechanism between Wt1 loss and mitotic arrest, we identified four potential WT1 binding sites within the cyclin E1 2.8-kb promoter (Figure 2F). The luciferase activity of TM4 cell transfected with WT1A or B (-KTS) was increased approximately 7-fold compared with the control. In contrast, no difference was observed between the control and WT1C or D (+KTS)-transfected TM4 cells (Figure 2G). To determine the effect of WT1(-KTS) in regulating Cyclin E1 promoter, we created two types of mutations in WT1A [37, 38]. Mutants contained changes in the zinc finger region, such as R366C, H377Y, and R394W, failed to activate the Cyclin E1 promoter. In contrast, the mutants with mutations outside the zinc finger region, such as F154S and S273A, had transactivational potentials similar to that of wild-type WT1A (Figure 2H). Together, these results indicate that WT1 regulates the expression of Cyclin E1. Moreover, loss of function of Wt1 in GR somatic cells induced cell morphological changes characteristic of epithelial-mesenchymal transition, accompanied by a complete loss of the epithelial markers E-cadherin and a significant elevation of mesenchymal markers vimentin (Figure 2I). Taken together, these results indicate that the aberrant GR development in Wt1-mutant embryos is due to the mitotic arrest rather than apoptosis of coelomic epitheliums, and Wt1 is essential for Cyclin E1 regulation, epithelial cells characteristics maintenance and their normal proliferation.
The directional migration of PGCs is not obviously affected in GR-deficient embryos
The expression of Sdf1, Steel factor and Integrin-β1 was not changed in Wt1R394W/R394Wembryos
The proliferation of PGCs in the developing gonad is regulated by somatic cells
The PGC identity and PGC-PGC interactions are not changed in Wt1R394W/R394Wembryos
The PGCs are formed distantly from their final destination, thus, they must migrate for a long distance to reach the GR. The precise mechanism that regulates the directional migration of PGCs towards the GR remains an open question . The widely accepted hypothesis suggests that the PGCs are attracted by the factors that are emitted from the destination or by somatic cells along their migratory route. In vitro studies have demonstrated that the GR tissue from E10.5 embryos attracts PGCs migration . Several genes have been implicated in the process of PGC migration, such as Sdf-1, Steel factor, Integrinβ1 [8, 15] and E-cadherin , and the inactivation of these genes results in aberrant PGC migration. In this study, a GR-deficient mouse model, Wt1R394W/R394W, was used to study the roles of the GR in germ cell migration and development. We found that the migration of PGCs in GR-deficient embryos was normal, which was consistent with a previous study . Stella-positive PGCs reached the mesenchyme under the coelomic epithelium at E10.5, and no ectopic PGCs were observed in GR-deficient embryos. However, the number of PGCs was dramatically reduced in GR-deficient at E11.5 and E12.5. Further study revealed that the decrease of PGC number was due to the reduced proliferation but not cell apoptosis.
Sdf1, Steel factor and Integrinβ1 have been reported to play important roles in regulating PGC migration [8, 15]. In this study, we found that the expression of these proteins was not restricted to GR somatic cells and the protein level was also not changed in Wt1R394W/R394W embryos compared to control embryos. These results suggest that the signals that regulate PGC migration probably not only come from GR somatic cells, but the factors from hindgut, mesentery and the mesenchymal cells around GR also play important roles in this process. Given that PGCs begin to migrate at approximately E7.5, while the GR is not visible until approximately E9.5 in mice, the early stage of PGC migration is most likely regulated by somatic cells along the migrating pathway, not by signals from the GR. In this study, we found that the GR development was blocked in Wt1R394W/R394W embryos due to Wt1 mutation. However, the coelomic epithelium was still maintained from E9.5 to E12.5. We could not exclude the possibility that the developmentally arrested coelomic epithelial cells still can secret some unknown factors which attract the PGC migration, and other functional GR is not essential for germ cell migration. We also found that most of the PGCs in control and Wt1R394W/R394W embryos arrived at the mesenchyme near the coelomic epithelium at E10.5. However, at E11.5 and E12.5, all the germ cells in control embryos were colonized into the developing GR (Figure 3B, C), whereas, the germ cells in GR-deficient embryos were scattered widely under the coelomic epithelium instead (Figure 3E, F). These results suggest that GR somatic cells are important for the precise positioning of PGCs at the final step of migration.
During and after colonization into the GR, PGCs proliferate rapidly, and their number is increased dramatically from approximately 500 to 25,000 by E13.5 [48, 49]. After sex determination, most germ cells enter mitotic arrest [20, 21]. Whether the proliferation of germ cells is a cell autonomous process or regulated by GR somatic cells is unclear. In this study, we found that the proliferation of PGCs was dramatically reduced in Wt1R394W/R394W embryos at E11.5 and E12.5 compared to control embryos, whereas, the germ cells in Wt1R394W/R394W embryos were still mitotically active at E13.5 when all the germ cells stop proliferation in control embryos, indicating that the proliferation of germ cells is precisely regulated by GR somatic cells during the early stage of gonad development, and disruption of GR development results in aberrant germ cell proliferation. Our results were consistent with previous in vitro studies, which showed that soluble factors released by GR regulated the proliferation of PGCs using the medium conditioned by different embryonic tissues .
The results of our study provide in vivo evidence that the proliferation of germ cells is tightly regulated by GR somatic cells during the early stage of gonad development. The directional migration of PGCs is not affected, but the precise positioning of PGCs at the final step of migration is disrupted in GR-deficient embryos, suggesting that the functional GR is not essential for PGC migration. However, the detailed molecular mechanisms underlying this regulation await further investigation.
All animal work was approved by the committee on animal care at the Institute of Zoology of the Chinese Academy of Sciences. All mice were maintained in a C57BL/6; 129/SvEv mixed background. Wt1R394W/R394W mice were obtained by crossing male and female Wt1+/R394W mice . DNA isolated from adult tails and fetal tissues was used for genotyping. Genotyping was performed by PCR as described previously . The sex of the embryo was detected by PCR using Sry primer .
Immunohistochemistry and immunofluorescence
Embryos were collected at E9.5 to E13.5. Embryos were dissected in PBS and fixed in 4% paraformaldehyde for up to 24 hours, stored in 70% ethanol, and embedded in paraffin. Tissue sections (5 μm thick) were cut and mounted on glass slides. Sections were deparaffinized and rehydrated, followed by antigen retrieval in 10 mM sodium citrate buffer. After blocking with 5% BSA for one hour, the sections were incubated with primary antibody at 4°C overnight. After washing with PBS, secondary antibody was applied for one hour, followed by washing in PBS. Staining was visualized using a DAB substrate kit (Zhong Shan Technology, Beijing, China). For immunofluorescence, the sections were blocked in blocking buffer (goat serum, 0.3% Triton X-100 in PBS) at room temperature for one hour and then incubated with primary antibodies overnight at 4°C. Sections were washed in 0.3% Triton X-100 in PBS and incubated with FITC or TRITC-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for one hour. The sections were washed in 0.3% Triton X-100 in PBS and counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma, St. Louis, MO, USA) to identify the nuclei.
The following antibodies were used in this study: rabbit anti-WT1 (1/400, Epitomics, Burlingame, CA, USA, 2797-1), rabbit anti-Stella (1/200, Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-67249), rabbit anti-Ki67 (1/500, Abcam, Cambridge, MA, USA, ab15580), mouse anti-Dazl (1/400, AbD Serotec, Raleigh, NC, USA, MCA2336), goat anti-Steel (1/50, Santa Cruz Biotechnology, sc-1303), rabbit anti-Sdf1 (1/50, Santa Cruz Biotechnology, sc-28876), rabbit anti-Integrinβ1 (1/500, Chemicon, Billerica, MA, USA, AB1952), rabbit anti-E-cadherin (1/200, Abcam, ab15148), and rabbit anti-Vimentin (1/200, Cell Signaling Technology, Danvers, MA, USA, #3932).
Count PGC number
A serial section of the whole embryo was prepared and stained by PGC-specific marker Stella. The Stella+ cells were counted at each section and added to the total PGC number of that embryo.
TUNEL assays were conducted with the In Situ Cell Death Detection Kit, Fluorescein (Promega BioSciences, San Luis Obispo, CA, USA), as recommended. Images were acquired with a Nikon DMR Epifluorescence Microscope (Nikon Instruments Inc, Melville, NY, USA), and images were captured by a Hamamatsu CCD camera (Hamamatsu Photonics, Iwata, Shizuoka, Japan).
BrdU incorporation assay
BrdU labeling and detection were conducted as previously described . Briefly, pregnant females were injected with 50 mg/kg of BrdU (Sigma) (intraperitoneally, i.p.) one hour prior to embryo collection. Embryos were dissected and fixed in 4% PFA. A purified mouse anti-BrdU monoclonal antibody (BD Bioscience, San Jose, CA, USA) was used for BrdU detection.
The genital ridge were lyzed with Trizol reagent (Invitrogen, Carlsbad, CA, USA) and total RNA was extracted according to the manufacturer's instructions. Measurement of RNA integrity and cDNA synthesis of cDNA were performed as previously described . Each sample was measured in duplicate in at least three independent experiments. Sample CT values were normalized to the corresponding Gapdh or Stella CT values, and relative expression levels were calculated using the ΔΔCT method . Primer pairs were listed in Additional file 1, Table S1.
Plasmid construction and cell transfection
Mouse Wt1 cDNA was amplified by PCR using testis cDNA and subcloned into the PCB6+ vector, as described . The 2.8-kb cyclin E1 promoter fragment was amplified by PCR from mouse genomic DNA and subcloned into the pGL3-basic luciferase reporter vector (Promega). Point mutation of WT1A was performed by site-directed mutagenic PCR, using the PCB6+WT1A construct as the template . TM4 cells were grown in F-12/DMEM supplemented with 10% fetal calf serum and cells were co-transfected with expression and reporter plasmids and the Renilla luciferase reporter plasmid (pRL-TK) (Promega) as indicated in the figure legends. The cells were harvested for the luciferase assay 36 hours after transfection.
Experiments were repeated at least three times. The data were evaluated for significant differences using a Student's t-test. A P-value <0.05 (*) was considered significant. A P-value <0.01(**) was considered very significant. Bar graphs were plotted in MS Excel (Microsoft Corporation, Redmond, WA, USA).
Bone morphogenetic protein
Bovine serum albumin
Cycle-dependent kinase 2
Cyclin-dependent kinase inhibitor
Dulbecco's Modified Eagle Medium
Polymerase chain reaction
Primordial germ cells
- Sdf1 :
Stromal cell-derived factor 1
Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick endlabeling
Wilms' tumor 1
We thank Vicki Huff (MD Anderson Cancer Center) for the supply of Wt1+/R394W mice.
This work was supported by the National Key Basic Research Program of China (2013CB945001, 2011CB944300 and 2012CB944702), CAS Innovation Project (Grants KSCX2-YW-R-081), and National Natural Science Foundation of China (31071271, 31071018, 30618005 and 30230190).
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