Fanconi Anemia (FA) is an autosomal recessive disorder characterized by cellular hypersensitivity to DNA cross-linking agents. Recent studies suggest that FA proteins share a common pathway with BRCA proteins. To study the in vivo role of the FA group A gene ( Fanca ), gene-targeting techniques were used to generate Fancatm1Hsc mice in which Fanca exons 1–6 were replaced by a β-galactosidase reporter construct. Fancatm1.1Hsc mice were generated by Cre-mediated removal of the neomycin cassette in Fancatm1Hsc mice. Fancatm1.1Hsc homozygotes display FA-like phenotypes including growth retardation, microphthalmia and craniofacial malformations that are not found in other Fanca mouse models, and the genetic background affects manifestation of certain phenotypes. Both male and female mice homozygous for Fanca mutation exhibit hypogonadism, and homozygous females demonstrate premature reproductive senescence and an increased incidence of ovarian cysts. We showed that fertility defects in Fancatm1.1Hsc homozygotes might be related to a diminished population of primordial germ cells (PGCs) during migration into the gonadal ridges. We also found a high level of Fanca expression in pachytene spermatocytes. Fancatm1Hsc homozygous males exhibited an elevated frequency of mispaired meiotic chromosomes and increased apoptosis in germ cells, implicating a role for Fanca in meiotic recombination. However, the localization of Rad51, Brca1, Fancd2 and Mlh1 appeared normal on Fancatm1Hsc homozygous meiotic chromosomes. Taken together, our results suggest that the FA pathway plays a role in the maintenance of reproductive germ cells and in meiotic recombination.
Fanconi Anemia (FA) is an autosomal recessive chromosomal-instability disorder. Cells derived from FA patients are hypersensitive to DNA cross-linking agents, and clinical manifestations involve a diverse assortment of hematological deficiencies, developmental abnormalities, reproductive defects and cancer susceptibility ( 1 ). Hematological complications may vary from mild anemia to bone marrow failure. Birth defects frequently involve growth retardation, skeletal abnormalities, microcephaly, microphthalmia and other facial dysmorphias, abnormal skin pigmentation, and cardiac, renal or gastrointestinal malformations ( 2 ). Male FA patients have underdeveloped gonads and defective spermatogenesis. Female FA patients have hypoplastic ovaries and infantile uterus. Sexually mature female FA patients may have menstrual irregularities, secondary amenorrhoea, anovulatory periods, premature menopause and increased risk of gynaecologic malignancies, although they are able to establish and maintain pregnancy ( 3 ). FA patients also have a high propensity towards malignancy, especially acute myelogenous leukemia. Older FA patients are at an increased risk of developing solid tumors, the most frequent being squamous cell carcinoma ( 4 ).
Apart from DNA cross-linking agents, cells derived from FA patients are also hypersensitive to ionizing irradiation and oxygen radicals, treatments that lead to increased apoptosis or growth arrest ( 2 ). Compromised growth capacity is observed when FA hematopoietic cells are exposed to cytokines such as interferon-γ, tumor necrosis factor-α and mip-1α ( 5 ). In addition, FA cells exhibit a prolonged late S and G2/M phase of the cell cycle, damage-resistant DNA synthesis, deficient DNA end joining activity, elevated homologous recombination activity, and accelerated telomere shortening ( 6 , 7 ).
Somatic cell hybrid studies of FA cells demonstrate the existence of at least eight complementation groups. The disease causing genes for complementation groups A ( FANCA ), C ( FANCC ), D1 ( BRCA2 ), D2 ( FANCD2 ), E ( FANCE ), F ( FANCF ) and G ( FANCG ) have been cloned, and there is evidence that mutations in BRCA2 may also account for complementation group B ( 6 , 8 ). FANCA, C, E, F and G are components of a multi-protein nuclear complex required for the mono-ubiquitination of FANCD2 in response to DNA damage or during the S phase of the cell cycle ( 9 ).
Despite the cloning of FA genes, the precise role of the FA pathway is still largely unknown. Recent results suggest that FA may function in a common pathway with BRCA proteins. Activated FANCD2 is targeted to BRCA1 and RAD51 nuclear foci, while the formation of RAD51-containing foci seems to be impaired in some FA cell-lines ( 10 ). Furthermore, disruption of BRCA1 results in the loss of DNA damage-inducible FANCD2-containing subnuclear foci ( 9 ). The recent identification of BRCA2 as the gene mutated in FANCD1 cells reinforces this association further.
Complementation group A accounts for 65% of all FA patients. The FANCA gene encodes a protein of 1455 amino acids with a molecular mass of approximately 163 kDa ( 11 ). FANCA protein is detected both in the nucleus and cytoplasm, and deletion of the amino-terminal end abolishes nuclear localization and its ability to correct mitomycin C (MMC) sensitivity ( 12 ). In addition to being an integral part of the FA nuclear complex, FANCA directly interacts with other proteins including BRCA1, BRG1, sorting nexin 5 (SNX5) and IκB kinase-2 (IKK2) ( 13 – 16 ), and co-localizes to nuclear foci with xeroderma pigmentosum complementation group F (XPF) and non-erythroid alphaII spectrin ( 17 ). Furthermore, five FA proteins including A, C, E, F and G have been found to form a complex with topoisomerase IIIα (Topo IIIα), replication protein A (RPA) and the Bloom's syndrome protein (BLM) ( 18 ). FANCA thus plays a crucial role in linking the FA complex with other pathways.
The mouse Fanca homolog shares 65% amino acid identity with human FANCA and expression of the mouse cDNA in human FA-A cells restores the cellular drug sensitivity to normal levels, suggesting that the mouse will be a useful model for the study of FANCA in vivo ( 19 ). Here we describe the creation and phenotypic characterization of mutant mice that carry a homozygous germ-line deletion of the functionally important exons 1–6 of Fanca. Interestingly, these Fancatm1Hsc and Fancatm1.1Hsc homozygous mice display defects that resemble those observed in FA patients, some of which are not seen in other Fanca mouse models ( 20 , 21 ). We show that the penetrance of certain FA phenotypes is dependent on the genetic background. Using these mice, we provide evidence that Fanca is involved in PGC survival or proliferation, and in the regulation of meiotic recombination.
Generation and characterization of Fanca mutant mice
We replaced exons 1–6 of Fanca with a β- galactosidase reporter by homologous recombination in murine embryonic stem cells (Fig. 1 A). Since genetic background and the presence of a neomycin selection cassette have been shown to affect the phenotype of genetically engineered animals, we subsequently generated a Fancatm1.1Hsc line with the neomycin cassette removed to evaluate the phenotypic consequences from these modifications (see Materials and Methods for details) ( 22 , 23 ). We also assessed offspring arising from successive generations of Fancatm1Hsc homozygous intercrosses in the 129S6 background for any differences in phenotype.
Heterozygous matings produced viable Fancatm1Hsc and Fancatm1.1Hsc homozygous mice with the expected Mendelian frequency (Fig. 1 B). No Fanca transcripts were detected in the testes of both Fancatm1Hsc and Fancatm1.1Hsc homozygous mice by RT–PCR using primers amplifying exons 6–9, although transcripts were detected with primers amplifying exons 14–18 (Fig. 1 C), suggesting that although the knockout strategy was successful, Fanca transcription reinitiated downstream. We were unable to find antibodies that recognize the C-terminal region of mouse Fanca, therefore we do not know whether this truncated transcript encodes a partial protein.
Phenotypic abnormalities in Fanca mutant mice
Fancatm1.1Hsc homozygous mice in the C57BL/6 background weighed less than their heterozygous littermates at birth, at weaning and as adults, suggesting that growth retardation occurred prenatally and persisted into adulthood (Fig. 2 A). When bone marrow cells from Fancatm1.1Hsc heterozygotes and homozygotes in the C57BL/6 background were cultured in methylcellulose with increasing concentrations of mitomycin C (MMC), progenitors from Fancatm1.1Hsc homozygotes had a significantly lower survival rate compared with those from heterozygote marrow, indicating that Fancatm1.1Hsc homozygotes mimic FA patients in their increased hypersensitivity to DNA cross-linking agents (Fig. 2 B).
FA is associated with an increased predisposition to cancer ( 4 ). We therefore monitored tumor formation in Fancatm1Hsc mice in the 129S6;CD-1 mixed background and the 129S6 inbred background. While no tumors were found by gross observation in wildtype and heterozygotes, we detected tumors in 33% (2/6) of Fancatm1Hsc homozygous mice in the 129S6;CD-1 background and 11.7% (2/17) of Fancatm1Hsc (G3) homozygous mice in the 129S6 background at 15 months of age (Table 1 ). Although the differences in tumor incidence were not statistically significant due to small sample sizes, it was notable that all tumors were found in homozygous mice.
While no macroscopic developmental abnormalities were observed with mutant mice in the 129S6 background, there was a significant increase in the incidence of microphthalmia in Fancatm1.1Hsc homozygotes as compared with Fancatm1.1Hsc heterozygotes and wildtype in the C57BL/6 background. We examined 27 litters of heterozygote intercrosses, and found that 0 of 50 wildtype, 2 of 106 (1.9%) heterozygotes and 10 of 33 (30.3%) homozygotes developed microphthalmia ( P <4.0E−09). These mice with eye defects frequently exhibited varying degrees of craniofacial abnormalties as well, mostly involving the rostrum that deviated dextrally (Fig. 2 C). Newborn pups were doubly stained by Alizarin red and Alcian blue to evaluate the development of the skeletal system. With the exception of an overall reduction in size, we did not find any obvious malformations of the skeleton in Fancatm1.1Hsc homozygotes in the C57BL/6 background (Fig. 2 D).
Necropsy analysis of Fancatm1Hsc and Fancatm1.1Hsc homozygous mice did not reveal any significant findings with the exception of smaller testes and ovaries. While 10-week-old heterozygous mice had similar testicular weights as wildtype mice, the testicular weights of Fancatm1Hsc homozygotes from a 129S6 background (0.048 g, n =12) were approximately 2-fold less than heterozygotes of the same background (0.104 g, n =10). Interestingly, the testicular weights of Fancatm1.1Hsc homozygotes in a C57BL/6 background were significantly reduced (0.026 g, n =8) compared to Fancatm1Hsc homozygotes from a 129S6 background ( P =8.7E−06) or heterozygotes of the C57BL/6 background (0.111 g, n =6).
We compared Fancatm1Hsc and Fancatm1.1Hsc homozygous mutant mice in the C57BL/6 background, and did not find any significant difference in the body weight, testis weight, or frequency of microphthalmia and craniofacial abnormalities, suggesting that the observed phenotypes are due to defective Fanca function manifested in the C57BL/6 background, and not to inadvertent complications resulting from the targeting strategy or the expression of neomycin.
Gonadal defects in Fanca mutant mice
Histological analysis of the testes from 10-week-old Fancatm1Hsc and Fancatm1.1Hsc homozygotes revealed a mosaic pattern with some tubules showing relatively normal spermatogenesis, while others were characterized by marked degeneration with dysplastic and degenerate tubular cells, absent or flattened Sertoli cells and scattered multinucleated giant cells and cellular debris within the tubules (Fig. 3 A and B). Multifocal to diffuse hypertrophy of the interstitial (Leydig) cells was also observed. Consistent with the difference in testicular weight, only ∼3% of the tubules in Fancatm1Hsc homozygotes of a 129S6 background were severely degenerated, compared to 68% of the tubules in Fancatm1.1Hsc homozygotes of a C57BL/6 background ( n =3, P =0.002).
Histological analysis of ovaries from 12-week-old homozygous females showed ovarian hypoplasia, characterized by reduced ovarian size, prominent epithelial cords, small degenerate follicles and abundant interstitium (Fig. 3 C and D). The number of follicles was markedly reduced compared to normal control tissue, although morphologically normal oocytes at all stages of oogenesis and corpora lutea were present.
We evaluated the reproductive performance of Fancatm1Hsc homozygotes by monitoring the number of pups born per litter over time and found that the fertility of Fancatm1Hsc homozygotes in the 129S6 background was similar to previously published results ( 20 ). Homozygous females in the 129S6 background exhibited significantly reduced litter sizes and an earlier onset of reproductive senescence, while the fertility of 129S6 homozygous males was not significantly different from heterozygotes (data not shown). Successive intercrossing of Fancatm1Hsc homozygous mice in the 129S6 background over three generations did not have a significant effect on fertility. However, preliminary breedings of homozygous Fancatm1.1Hsc mice in the C57BL/6 background did not produce offspring (data not shown).
We examined the ovaries from Fancatm1Hsc homozygotes histologically at 7 months to determine the cause of infertility. No follicles were observed in the ovaries, concomitant with the appearance of hemorrhagic or fluid-filled cystic structures, suggesting that the reason for premature onset of infertility was due to a depletion of oocytes (Fig. 3 E and F). Examination of the ovaries in 15-month-old females showed that only 0 of 4 (0%) wildtype and 1 of 6 (16.7%) heterozygotes developed cysts. Meanwhile, 7 of 9 (77.8%) homozygous ovaries were cystic, and ovaries from the same animal frequently showed divergent morphologies ( P <0.003) (Fig. 3 G). An ovarian granulosa cell tumor was found in a 17-month-old Fancatm1Hsc (G3) mouse (Table 1 ).
To investigate the onset of germ cell defects, we stained E15.5 fetal ovaries and newborn testes with the germ cell-specific marker Gcna-1. We found that fetal ovaries and newborn testes from Fancatm1.1Hsc homozygotes had significantly fewer germ cells compared with their heterozygous littermates, suggesting that a prenatal defect in germ cell development affected both males and females (Fig. 3 H–K).
Analysis of PGCs in Fanca mutant mice
All gametes are derived from primordial germ cells (PGCs). In the mouse, PGCs can be first seen at the midgastrula stage (E7.25–E7.75) in the extraembryonic mesoderm posterior to the primitive streak ( 24 ). PGCs migrate from the base of the allantois at E8.5 to the genital ridge at E11.5. To determine the stage of germ cell reduction in Fancatm1.1Hsc homozygotes, we quantitated the number of PGCs in E8.5 and E11.5 embryos. We took advantage of the fact that PGCs have high alkaline phosphatase content, which can be detected by Fast Red staining ( 24 ). At E8.5, no significant difference in the number and location of primordial germ cells was found between control and mutant mice (Fig. 4 A and B), suggesting that the defect was not in the establishment of the founder population of PGCs. By E11.5, Fancatm1.1Hsc homozygotes contain approximately 50% of the number of PGCs in control embryos, and examination of E11.5 embryos did not reveal any ectopically located PGCs, suggesting that the primary defect was not in migration, but most likely in the survival or proliferation of PGCs (Fig. 4 C–E).
Expression of Fanca in mice
As the β-galactosidase gene was knocked-in under the control of the Fanca promoter, we tested if β-galactosidase activity served as a marker for Fanca expression. Homogenates of tissues from 10-week-old wildtype and Fancatm1.1Hsc heterozygous mice were used in a luminescence-based assay to examine the level of β-galactosidase activity, and we found an ∼400-fold increase in β-galactosidase activity in the testes from heterozygotes relative to wildtype controls (Fig. 5 A). We also examined Fanca expression during embryonic development by staining E7.5–E13.5 embryos, as well as fetal gonads from E14.5 to E17.5, in X-gal. However, no blue staining was observed in heterozygous or homozygous embryos (data not shown).
We confirmed Fanca expression in the testis by northern analysis. An intense 4.4 kb band representing full-length Fanca was detected only in RNA isolated from the wildtype testis, although equal quantities of testis and spleen RNA were loaded (Fig. 1 D). To further determine the cell type of Fanca expression in the testis, we examined β-galactosidase activity in testis lysates from prepubertal mice. Since the first round of spermatogenesis is synchronous, we can determine if Fanca expression is temporally linked to the appearance of specific germ cell types by monitoring the time of increased β-galactosidase activity ( 25 ). No significant β-galactosidase activity was detected until day 18, which coincided with the emergence of mid- to late-pachytene spermatocytes (Fig. 5 B). The expression pattern of Fanca in the testis was further examined with in situ β-galactosidase staining in intact seminiferous tubules and testis sections from 10-week-old mice. In spermatogenesis, distinct association of germ cell types at specific phases of spermatogenic development are known as stages, and a particular ordering of segments at the same stage occur along the length of the seminiferous tubule ( 26 ). Blue staining was observed in segments of the seminiferous tubules in Fancatm1.1Hsc heterozygotes in a periodic pattern, suggesting that the expression was controlled in a stage-specific manner (Fig. 5 C–E). X-Gal staining in testis sections confirmed that strong β-galactosidase activity was detected specifically in the nucleus of late-pachytene spermatocytes at stages VIII–X, but not in the earlier stages (Fig. 5 F–H). No significant difference was observed in the level and location of β-galactosidase activity whether or not the neomycin cassette was present. To confirm whether X-gal staining reflected the cells in which Fanca was expressed, we performed in situ hybridization of Fanca on 10-week-old wildtype mouse testis and found a more intense signal in the spermatocytes of some tubules (Fig. 5 I–K). Taken together, these analyses showed that Fanca expression peaked during late-pachytene.
Spermatogenesis in Fanca mutant mice
Since stage-specific Fanca expression was detected in the testis, we further investigated spermatogenesis in 10-week-old Fancatm1Hsc homozygous males in the 129S6 background. Germ cells in homozygous males appeared to be actively proliferating, as most of the spermatogonia and spermatocytes stained positive for the cell proliferation marker PCNA (proliferating cell nuclear antigen) (Fig. 6 A and B). However, there appeared to be fewer proliferating cells per tubule, due to the depletion of spermatogonia and spermatocytes, despite a relatively normal number of spermatids. TUNEL analysis showed a significant increase in the frequency of tubules with apoptotic cells in Fancatm1Hsc homozygotes of the 129S6 background (Fig. 6 C). The apoptotic cells in homozygous mice were located close to the basal membrane, suggesting that they are mostly spermatocytes (Fig. 6 D).
Since Fanca expression was developmentally regulated in cells undergoing meiotic recombination, we examined meiotic progression in 3-week-old Fancatm1Hsc mice of the 129S6 background by staining the chromosome axes using an Scp3 antibody. In pachytene spermatocytes taken from three homozygotes, we found that 11.4% (28 of 245) showed mispairing (Fig. 6 E), compared to 2.3% (4 of 175) from two heterozygotes and 3.5% (2 of 58) analyzed in one wild-type mouse ( P <0.0009).
In mitotic cells, BRCA1, BRCA2 and RAD51 interact and co-localize in a punctate pattern in the nucleus during the S phase of the cell cycle, whereas in meiotic cells, all three proteins associate with unsynapsed chromosome axes along the developing synaptonemal complexes (SCs) ( 27 ). Since previous reports suggested that the absence of FA proteins may impair the formation of DNA damage induced RAD51, FANCD2 and BRCA1 foci in mitotic cells, we examined whether the chromosomal association of Rad51, Fancd2 and Brca1 was affected in spermatocytes from Fancatm1Hsc homozygotes ( 28 , 29 ). Similar to published results, Rad51 was localized in discrete foci along the unsynapsed and synapsed chromosome axes from leptotene to early pachytene. The number of Rad51 foci reached a maximum at leptotene, and declined to zero by mid or late pachytene in most nuclei. Interestingly, an abundant number of Rad51 foci remained on the mispaired axes during pachytene (Fig. 6 E). Fancd2 formed discrete foci along the synapsed axes in pachynema (Fig. 6 F), and specifically stained the unpaired sex chromosomes in late pachynema. Similar to Fancd2, we also observed specific Brca1 staining of the unpaired sex chromosomes in pachynema (Fig. 6 G). The number, intensity and temporal pattern of these foci appeared similar to heterozygous controls.
To investigate a later stage at meiotic recombination, we examined the chromosomal location of Mlh-1 in spermatocytes from Fancatm1Hsc homozygotes (Fig. 6 H). Mlh-1 is a mismatch-repair protein that localizes on meiotic chromosomes at discrete foci representing late recombination nodules, and is thought to mark the sites of crossing over ( 30 ). No abnormality was detected in Mlh-1 localization, and from 27 Fancatm1Hsc homozygous pachytene nuclei that had 19 or more Mlh-1 foci, we calculated an average of 22.5 Mlh-1 foci per nuclei. This value is very close to the published data, suggesting that the absence of Fanca does not affect recombination frequency.
Novel phenotypes in Fanca mutant mice
To examine the role of Fanca in normal growth and development, we have generated Fancatm1Hsc and Fancatm1.1Hsc mice by replacing the first 6 exons of the Fanca gene with a β- galactosidase gene. In addition to the previously reported hypogonadism and MMC hypersensitivity, Fancatm1.1Hsc homozygous mice show additional FA-like phenotypes that are not reported in other published Fanca mouse models, including growth retardation, microphthalmia and craniofacial defects. Interestingly, growth retardation, germ cell defects and DNA repair deficiency are also phenotypes found in mice which have partial loss of Brca gene function, suggesting that Fanca may participate in some, but not all, Brca functions ( 31 , 32 ).
Both the specific gene mutation and the genetic background have been demonstrated to affect the clinical manifestation in FA patients, and it is possible that these factors may contribute to the phenotypic differences we observe in our model versus previously published models ( 33 ). We designed our targeting strategy to disrupt the amino terminal region of Fanca, whose corresponding region in human FANCA was known to be important for nuclear translocation, as well as interaction with FANCG and BRCA1 ( 12 , 13 , 34 ). In addition, we analyzed the mutants in two different genetic backgrounds. When we compared Fancatm1Hsc homozygotes in the 129S6 and C57BL/6 backgrounds, we found a more severe depletion of germ cells in Fancatm1Hsc homozygous males when they were in the C57BL/6 background, indicating that genetic background affected the penetrance of the FA phenotype in mice.
More interestingly, Fanca also acts as a modifier gene. C57BL/6 mice have a genetic predisposition for microphthalmia, which is related to developmentally retarded lens development ( 35 ). In this study, we show that Fancatm1.1Hsc homozygosity enhances the occurrence of microphthalmia in C57BL/6 mice. The frequency of ocular abnormalities in C57BL/6 mice can also be enhanced by environmental influences such as alcohol exposure, or by genetic modifiers such as p53, both of which are associated with the control of apoptosis ( 36 , 37 ). Based on these results, we propose that Fanca does not play a direct role in the formation of the eyes, but rather, its involvement in the control of cell proliferation and apoptosis may affect embryonic development. Our hypothesis helps explain why heterogeneous organ pathologies have been reported between FA patients with the same mutation ( 38 ).
Reproductive defects in Fanca mutant mice
Reproductive defects are the most consistent phenotype seen in FA mouse models, and our study suggests that the combination of PGC reduction and meiotic defects may have caused the problem ( 39 ). We have determined that the germ cell defect in Fancatm1.1Hsc homozygous mice initiates between E8.5 and 11.5, when PGCs leave their extraembryonic site, proliferate and migrate along the developing hindgut toward the urogenital ridges. Since no migration defect has been observed, the primary defect is most likely in the survival or proliferation of PGCs. Our observation is consistent with that found in Fancc−/− mice, where there is a severe reduction of germ cells at E12.5 compared with control littermates ( 40 ).
Fanca has been shown to express in E7.5 and E11.5 embryos by northern analysis ( 19 ), and we have confirmed the expression of Fanca in E8.5 and E10.5 embryos by RT–PCR (data not shown). Fanca was also found to be expressed in E11.5 genital ridges by RT–PCR ( 40 ). However, we failed to detect β-galactosidase activity in embryos at these stages, and previously published in situ hybridization results only detected a diffuse but not localized expression of Fanca at E11.5 ( 41 ). Therefore, we were unable to determine the expression pattern of Fanca in PGCs.
To date, only a handful of genes have been identified which affect PGC proliferation in vivo between E8.5 and E11.5. Mutant mice lacking TIAR (T-cell-restricted Intracellular Antigen-related Protein) or Pog gene (proliferation of germ cells) both exhibit reduced body weight, strain dependent partial embryonic lethality and adult sterility ( 42 , 43 ). However, the role of these genes on PGC survival and proliferation has not been well characterized. In contrast, W and Sl mutants involving the genes that encode tyrosine kinase receptor c-Kit and its membrane bound ligand Scf (stem cell factor) are better characterized. Decreased PGC migration and reduced proliferation are associated with the W and Sl mutants. Studies on cultured PGCs demonstrate that the c-kit/SCF interaction is crucial for PGC survival, and SCF stimulates PGC proliferation ( 44 ). Apart from sterility, the W and Sl mutations also cause anemia and lack of pigment cells ( 45 ). Interestingly, defects in hematopoiesis and melanogenesis are also implied in FA patients. Furthermore, bone marrow cultures of Fanca−/− homozygous mice were found to have impaired ex vivo expansion capacity and increased apoptosis when stimulated with stem cell factor plus interleukin-11 ( 46 ). It is possible that the deregulated response to SCF may be extended to the germ cell setting, causing impaired proliferation and apoptosis in Fanca−/− PGCs.
Defective meiosis in Fanca mutant mice
In addition to PGC maintenance, we also provide evidence that Fanca plays an important role in meiosis. We found an increased incidence of mispaired chromosomes in pachytene spermatocytes, accompanied by increased male germ cell apoptosis in Fancatm1Hsc homozygous mice. Our analysis also implies that Fanca expression is tightly regulated during the meiotic phase of mouse spermatogenesis. At least two lines of evidence suggest that the β-galactosidase activity is a true representation of the in vivo expression of Fanca in the testis. First, the level of β-galactosidase activity is significantly higher in the testis compared with other tissues, which is consistent with the result from northern analysis. Second, in situ hybridization of Fanca on testis sections confirms the elevated level of Fanca expression in spermatocytes.
According to our results, an abrupt increase in Fanca expression is restricted to mid- to late-pachytene spermatocytes. This is the stage when chromosomes are fully synapsed, and when Holliday junctions are formed and resolved into recombinants. Meiotic recombination results from formation and repair of DNA double-stranded breaks, and many proteins involved in DNA repair also play critical roles in meiotic recombination ( 27 ). Furthermore, the repair of DNA interstrand cross-links is believed to involve the formation of double-stranded breaks and homologous recombination ( 47 ). Since the most consistent FA phenotype has been the sensitivity to DNA cross-linking agents, it is possible that the fundamental role of the FA pathway is in the regulation of homologous recombination.
The FANCA interacting proteins BRCA1 and XPF are known to be involved in homologous recombination. Similar to Fanca , Brca1 and Xpf also show elevated expression in pachytene spermatocytes, suggesting that they may function with the FA pathway to regulate meiotic recombination ( 48 , 49 ). In addition, Fancd2 has been found to co-localize with Brca1 in meiotic chromosomes, further supporting the view that FA and BRCA proteins cooperate during meiosis ( 9 ). However, we found that Fancd2, Brca1 and Rad51 were localized to the expected positions on SC even in the absence of a functional Fanca protein, indicating that Fanca was not necessary for the recruitment of these proteins onto meiotic chromosomes.
Most mouse models with meiotic defects demonstrate meiotic arrest ( 50 ). In male mice that carried a homozygous deletion of Brca1 exon 11 and a p53 heterozygous mutation, spermatogenesis failed to advance beyond the pachytene stage, while diminished Rad51 foci and absence of Mlh1 foci were observed in pachytene spermatocytes ( 51 ). In contrast, a proportion of spermatocytes in Fancatm1Hsc homozygous males progressed through spermatogenesis and produced functional spermatozoa, and we observed normal numbers of Rad51 and Mlh1 foci in Fancatm1Hsc homozygous pachytene spermatocytes, suggesting that the meiotic dysfunction resulting from loss of Fanca had a less severe impact on spermatogenesis than the loss of Brca1.
Based on the observation that Fanca expression was elevated during pachytene, and there was an increased number of mispaired chromosomes in Fancatm1Hsc homozygous pachytene spermatocytes, we speculate that the role of Fanca may be related to the resolution of defective recombinants. Interestingly, FANCA has very recently been found to associate in a complex with RPA and the BLM helicase, both of which also localize onto meiotic chromosomes during prophase I ( 52 ). Furthermore, despite a report documenting a marked increase in homologous recombination in FA cell extracts, we found no difference in the number of chiasmata in Fancatm1Hsc homozygous spermatocytes based on Mlh1 localization ( 53 ). This observation is consistent with a previous report showing that Blm deficiency increases the level of mitotic recombination, while meiotic recombination remains unaffected ( 54 ). Further investigations into the relationship between BLM, RPA and FANCA should aid in clarifying the role of Fanca during meiotic recombination.
Finally, we observed an increased occurrence of cystic ovaries in Fancatm1Hsc homozygous females. Similar ovarian phenotypes have been observed in other mouse models with an early depletion of maturing follicles, including Msh5 (MutS homolog 5) and Msh4 (MutS homolog 4) mutants which have defective meiosis, or W (white spotting) and Sl (Steel) mutants which have PGC depletion ( 55 – 58 ). Since oocytes secrete signals that induce follicle formation, prompt granulosa cell proliferation and regulate steroidogenesis, it is possible that the development of ovarian cysts and tumors may be a secondary defect due to the absence of functional oocytes ( 59 , 60 ).
We have developed a Fanca mouse model that shows novel phenotypes similar to FA patients, including growth retardation, microphthalmia and craniofacial defects. The penetrance of certain phenotypes is strain-dependent, suggesting that epistatic influences should be taken into account when studying Fanca. Using this mouse model, we identify Fanca as one of the few genes known to affect PGC maintenance during migration into the genital ridges, and provide evidence supporting a functional role for Fanca in meiotic recombination. Our findings document the utility of Fancatm1Hsc mice as an in vivo model for the study of FA.
MATERIALS AND METHODS
Generation of mutant mice
The genomic region of Fanca was isolated as described ( 19 ). Restriction mapping and partial sequencing was used to determine the structure of the locus as shown in Figure 1 A. A 5.2 kb EcoR I- Sma I fragment spanning the region from upstream of exon 1 to 46 bp after the putative ATG start codon was used as the 5′ homology arm of the targeting construct. An internal ribosomal entry site (IRES)—nuclear localization signal (NLS)— lacZ reporter construct (gift from A. Nagy) was inserted downstream of the 5′ arm, followed by a PGK-neo positive selection cassette flanked by loxP sites (gift from B. Hug). A 3.5 kb Sac I fragment containing exons 7–8 of Fanca was ligated downstream to the neo selection cassette as the 3′ homology arm.
The Xho I–linearized targeting vector was electroporated into TL1 embryonic stem cells (gift from P.A. Labosky) and selected for neomycin resistance according to standard protocols ( 61 ). Genomic DNA from resistant clones was digested with EcoR I and Southern blot analysis was performed using the 276 bp 3′ external Pst I probe as shown in Figure 1 A. Ten homologous integrants were identified, and the integrity of the 5′ end of the locus was confirmed by PCR using the primers Fanca-a (5′-AGC CGA TGT TCC AGA CGC TAT GC-3′), Fanca-b (5′-GGT ATC TCA GGA GTT TCA GAG CAG AAT CC-3′), and Fanca-c (5′-GCT TCC AGA GGA ACT GCT TCC TTC ACG-3′) as shown in Figure 1 A. PCR with Fanca-a and Fanca-b gives a 480 bp product diagnostic of the wild-type allele, whereas PCR with Fanca-a and Fanca-c gives a 316 bp fragment diagnostic of the targeted allele (Fig. 1 B). PCR conditions were 94°C for 5 min (one cycle), 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min (35 cycles) and a final extension of 72°C for 10 min (one cycle).
ES cells from three independently targeted cell lines were aggregated with CD-1 embryos and transferred into pseudopregnant CD-1 females as described ( 62 ). Agouti male chimeras were mated with CD1 or 129S6 females, and offspring were analyzed by Southern blot for the presence of the targeted allele. Two of the three targeted cell lines transmitted the mutation through the germline, and heterozygotes were interbred to obtain homozygotes (Fig. 1 B). The targeted allele is designated Fancatm1Hsc , according to the guidelines of the International Committee on Standardized Genetic Nomenclature for Mice (The Jackson Laboratories). Mice derived from both cell lines showed the same phenotype, and were routinely typed by PCR on tail tip DNA using the same protocol as described for ES cell analysis.
Fancatm1Hsc heterozygotes were mated with pCX-NLS-Cre transgenics (gift from C.C. Hui) to remove the neomycin cassette ( 63 ). Offspring positive for the targeted Fanca allele and loss of neomycin were bred with wild-type C57BL/6 mice, and were genotyped for the presence of the targeted Fanca allele and the loss of the Cre transgene. Detailed procedures for the genotyping are avaliable upon request. Mice carrying the targeted Fanca allele without neomycin were designated Fancatm1.1Hsc .
Mouse strains and mating scheme
Fancatm1Hsc mice were maintained on a 129S6 background, and were used for northern blots, RT–PCR, immunostaining, TUNEL analysis, meiotic chromosome staining and tumorigenesis evaluation in this study. Heterozygotes were intercrossed to obtain generation 1 (G1) homozygotes. Offspring from Fancatm1Hsc (G1) homozygous intercrosses generated Fancatm1Hsc (G2) animals. Following this mating scheme, Fancatm1Hsc homozygotes up to the fourth generation (G4) were produced. Fancatm1.1Hsc mice were maintained by serial backcrossing of heterozygotes to C57BL/6 wildtype mice, and heterozygotes from the fourth and fifth backcross generations were interbred to produce mice for body weight analysis, skeletal staining, β-galactosidase studies, primordial germ cell analysis and hematopoietic colony assays. For timed matings, detection of the vaginal plug was designated as E0.5. Date of birth was counted as day 0.
Northern, RT–PCR and in situ hybridization
We isolated total RNA from the testes and spleens of 10-week-old mice using TRIZOL reagent (Invitrogen). Northern analysis was performed as previously described, with the exception that 30 µg of total RNA were loaded per lane ( 19 ). For RT–PCR of exons 6–9 and exons 14–18 of Fanca, 1 µg of total RNA was amplified using the SUPERSCRIPT One-Step RT-PCR with Platinum Taq system (Invitrogen) using primers Fanca 541–559 for (5′-GTT GCT GGA AGC TAT GTG G-3′) and Fanca 830–812rev (5′-GCA TCC TCT GCA GTA CAT C-3′) or Fanca 1293–1317 for (5′-CTG CGT TCC TGA TCG TGC GCC AGG C-3′) and Fanca 1642–1621rev (5′-GCT CTC ACG CTC GGC AAT GTC C-3′), respectively. G3PDH was co-amplified as a positive control, using primers 5′G3PDH (5′-TCC ACC ACC CTG TTG CTG TAG-3′) and 3′G3PDH (5′-GAC CAC AGT CCA TGC CAT CAC T-3′). One cycle at 50°C for 30 min was used for the amplification of cDNA, followed by 94°C for 2 min (one cycle), 94°C for 15 sec, 58°C for 30 sec, and 72°C for 1 min (35 cycles) and a final extension of 72°C for 10 min (one cycle). In situ hybridization was carried out on frozen sections as described, using as a probe 1–725 bp of the mouse Fanca cDNA and hybridized overnight at 50°C ( 64 ).
Mice were monitored for tumors weekly up to 15 months, when the surviving animals were killed and subjected to necropsy. Fancatm1Hsc (G3) animals were permitted to survive beyond the 15 month endpoint, and the mean age of the cohort was 15 months (range 14–17 months) when this paper was written. Moribund animals or animals developing overt tumors before this endpoint were also killed and necropsied. Tumors were confirmed and categorized by histopathological evaluation.
Hematopoietic colony assay
Bone marrow cells were isolated from femurs and tibiae of 8-week-old mice, and 2×10 4 cells were cultured in 1 ml of Methocult M3434 media following standard protocols with or without MMC treatment (StemCell Technologies). Colonies were scored at day 7. Each number was averaged from duplicate plates and the data was derived from three independent experiments.
Whole mount skeletal preps
Newborn mice were stained with Alcian blue and Alizarin red and cleared as described ( 65 ).
β-Galactosidase assay and staining
Tissue lysates were assayed for β-galactosidase activity using the Galacto-Light Plus™ chemiluminescent reporter gene assay systems (Applied Biosystems). Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories). Intact seminiferous tubules were isolated as described ( 66 ). β-Galactosidase staining was performed as described ( 67 ). For staging, tubules were post-fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned and counterstained with periodic acid Schiff according to standard protocols.
Histology, immunohistochemistry and TUNEL analysis
Tissues were fixed in 10% neutral buffered formalin, paraffin embedded and sectioned at 5 µm. Routine haematoxylin and eosin staining was performed. Sections were prepared for immunohistochemistry according to the manufacturer's recommendation (Zymed). PCNA staining was performed using the PCNA staining kit (Zymed), and Gcna1 staining was performed with the Histomouse-SP kit (Zymed) using as a primary antibody undiluted rat hybridoma supernatant against germ cell nuclear antigen-1 (Gcna-1) (gift from G.C. Enders) and a biotinylated anti-rat IgG secondary antibody (Vector Laboratories). TUNEL analysis was performed using the In Situ Cell Death Kit, Fluorescein according to manufacturer's instructions (Roche).
Meiotic chromosome staining
Staining of meiotic chromosomes was carried out as described ( 9 ). For these studies, the following antibodies were used: RAD51, 1 : 100 (Oncogene); Brca1, 1 : 100 and FANCD2, 1 : 100 (gift from A.D. D'Andrea); SCP3, 1 : 100 (gift from J. Sweasy); and MLH1, 1 : 100 (BD Biosciences).
PGC staining and counting
PGC staining and counting were performed as described ( 68 ). For counting PGCs at E11.5, the genital ridges dissected from alkaline phosphatase-stained E11.5 embryos were embedded in OCT compound (Miles) and serially sectioned at 10 µm. Sections were collected on glass slides and mounted with GVA media (Zymed). Counting was performed on every fifth section under a light microscope.
Statistical analyses such as the Student's t -test or the Pearson's chi-square test were performed using Microsoft EXCEL software. Error bars indicate standard deviation. Differences were judged as significant if the P value was <0.05.
We thank A. Nagy, B. Hug, P.A. Labosky, C.C. Hui, G.C. Enders, A.D. D'Andrea and J. Sweasy for reagents; and L. Wei and L. Morikawa for technical help. We appreciate insightful comments on the manuscript from C.C. Hui and D.J. Stavropoulos. This work was supported by grants from the Canadian Institutes of Health Research and the National Cancer Institute of Canada, supported by the Canadian Cancer Society, and also by the Hospital for Sick Children Foundation. M.B. holds the Lombard Insurance Chair in Pediatric Research at HSC and the University of Toronto.
To whom correspondence should be addressed. Tel: +1 4168136361; Fax: +1 4168134931; Email: email@example.com
|Background||Genotype||Tumor type||No. of mice with tumors/analyzed|
|129 CD-1||HOMO||1 lymphoma||2/6 (33%)|
|1 malignant fibrous histiocytoma|
|129S6 (G3)||HOMO||1 thymic lymphoma||2/17 (11%) a|
|1 spindle-cell sarcoma|
|1 ovarian granulosa cell tumor b|
|Background||Genotype||Tumor type||No. of mice with tumors/analyzed|
|129 CD-1||HOMO||1 lymphoma||2/6 (33%)|
|1 malignant fibrous histiocytoma|
|129S6 (G3)||HOMO||1 thymic lymphoma||2/17 (11%) a|
|1 spindle-cell sarcoma|
|1 ovarian granulosa cell tumor b|
a 1 spontaneous death of unknown cause due to severe autolysis.
b This tumor was found in a mouse 17 months of age.