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Previous Article | Next Article 
Molecular and Cellular Biology, September 2006, p. 6557-6570, Vol. 26, No. 17
0270-7306/06/$08.00+0 doi:10.1128/MCB.00729-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Division of Developmental Biology,1 Radioisotope Experimental Laboratory, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan,2 Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan,3 Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan,4 Laboratory for Animal Resources and Genetic Engineering, RIKEN, Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan,5 REDS group, Saitama Small Enterprise Promotion Corporation, Skip City, Kawaguchi, Saitama 333-0844, Japan6
Received 27 April 2006/ Returned for modification 13 June 2006/ Accepted 20 June 2006
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ABSTRACT |
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INTRODUCTION |
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Somatic stem cells, including neural stem cells (NSCs) and hematopoietic stem cells (HSCs), share some of the properties of ESCs, including multipotency and self-renewal. In the event of severe injury, numerous types of tissue-specific stem cells can give rise to cells of heterologous lineages (39, 42, 43), although in some cases, fusion of stem cells with other cells appears to be involved in transdifferentiation (21, 29, 46). Thus, it is possible that ESCs and somatic stem cells share a common genetic program that maintains stem cell identity (20, 37, 40, 42). Recently, Ivanova et al. (13) identified 283 genes or expressed sequence tags, including a gene encoding junctional adhesion molecule B (JAM-B) (nomenclature of the protein in NCBI Database is JAM2), that are expressed in three different stem cell lines, by means of DNA microarray analysis. Although it is assumed that at least some of these genes are involved in the maintenance of stem cell properties, no data confirming this have yet been reported.
Here we investigate this possibility. We have focused on Jam-B, a putative "stemness" gene identified by Ivanova et al. (13), because of the extreme difference in expression levels between undifferentiated and differentiated ESCs. Jam-B encodes an immunoglobulin superfamily protein that is specific to tight junctions and mediates cell-cell contacts between T cells and endothelial cells and many other systems (2-4, 9, 11, 16-18, 32). We first generated ESCs in which Jam-B was doubly targeted. We also generated Jam-B knockout mice by targeting disruption to examine the role of the Jam-B gene in maintenance of the stem cell state of NSCs and HSCs and in other aspects of development. These analyses revealed that Jam-B mutant ESCs are normal in morphology and retain pluripotency. Moreover, we found that Jam-B knockout mice were viable and indistinguishable from wild-type mice in appearance. Furthermore, we found that NSCs and HSCs recovered from Jam-B mutant mice are equivalent to those recovered from wild-type mice in the common properties of stem cells, such as multipotency. Unexpectedly, our analyses also revealed that Jam-B mutant male mice were also normal in spermatogenesis, although it has been assumed that the JAM-B protein present in Sertoli cells plays crucial roles in spermatogenesis by interacting with the JAM-C protein present in spermatids (11).
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture. ZHBTc4 (28), E14tg2A (38), and TT2 (45) embryonic stem (ES) cells were cultured as described previously (27). Differentiation of ZHBTc4 ES cells (feeder free) was done simply by adding tetracycline (1 µg/ml) to normal ES medium containing leukemia inhibitory factor when transferred to a new tissue culture dish (5 x 105 cells per 10-cm dish) and cultured as a monolayer for 48 h.
Neurosphere culture. Forebrain cells were prepared as described previously (23) from 12.5- or 14.5-days postcoitum (dpc) embryos obtained from intercrosses of wild-type or Jam-B homozygous mutant mice. After dissociation into a single-cell suspension, cells were seeded onto noncoated 10-cm dishes at a concentration of 1 x 105 to 2 x 105 cells/ml. Cells were cultured for 4 to 6 days with 1x B27 supplement (Invitrogen)-containing medium plus 20 ng/ml basis fibroblast growth factor (bFGF) and epidermal growth factor (EGF). For comparing efficiency of neurosphere formation between wild-type and Jam-B mutant mice, dissociated forebrain cells (see Fig. 6B) or primary neurosphere cells (see Fig. 6D, left panel) were seeded into 96-well plates at the indicated concentrations. After 6 to 10 days of culture, neurospheres were counted under a microscope. For differentiation of neurosphere cells, primary spheres were dissociated into single cells and then cultured on poly-L-ornithine and fibronectin-coated dishes in the absence of bFGF and EGF.
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Reverse transcription-PCR analysis. Hematopoietic cells were recovered from bone marrow, thymus, and spleen of C57BL/6 mice and fractionated using fluorescence-activated cell sorting (FACS) with antibodies against the cell surface markers indicated in Fig. 2C. Total RNA was isolated using ISOGEN-LS (Nippon Gene, Tokyo, Japan) and reverse transcribed using an oligo(dT) primer. After normalization of cDNA content using the glyceraldehyde-3-phosphate dehydrogenase cDNA level as a standard, semiquantitative PCR was carried out. ß-Actin was used as an internal control. The primer set sequences for Jam-B and ß-Actin cDNA are as follows: Jam-B, 5'-ACGAAGCTTTCAATATACGAATCAAAA-3' and 5'-CATGTTGAATTGCAGAAT TC-3'; ß-actin, 5'-GGTCAGAAGGACTCCTATGT-3' and 5'-ATGAGGTAGTCTGTC AGGTC-3'. Real-time PCR analyses shown in Table 2 were performed as follows. Total RNAs were prepared from various tissues of 8-week-old mice and embryos at variable stages and reverse transcribed using a random primer. These samples were used to examine the levels of gene expression of Jam-A, -B, -C, and ß-Actin by real-time PCR using TaqMan probes from Applied Biosystems.
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Teratoma formation. Jam-B mutant as well as wild-type ESCs (1 x 107 cells/0.1 ml phosphate-buffered saline) were independently injected subcutaneously in nude mice as described previously (27).
Immunohistochemistry.
For immunohistochemistry of teratoma, the tumors were recovered from nude mice and fixed with 4% paraformaldehyde. The tumors were steeped in 30% sucrose solution overnight and then embedded in optimal cutting temperature compound. The frozen sections (thickness, 10 µm) were incubated with anticytokeratin (mixture of clones C-11, PCK26, CY-90, KS-1A3, M20, and A53-B/A2; Sigma), -
-fetoprotein (clone MAB1368; R & D Systems), -cardiac troponin I (clone 19C7; Fitzgerald), or -MYOD (sc-780; SANTA CRUZ) antibody. Appropriate Alexa Fluor dye-conjugated secondary antibodies from Invitrogen were used to visualize immunostaining. For immunostaining of forebrain and spinal cord, the same procedures as described above were applied using developing brains from 12.5-dpc embryos and 14.5-dpc whole embryos, respectively. The frozen sections were incubated with anti-Nestin (clone RAT401; BD pharmingen), -MAP2 (clone HM-2; Sigma), or -phosphohistone H3 (clone 6G3; Cell Signaling Technology) antibody. Alexa-594 Fluor dye-conjugated antibodies were used as secondary antibodies. For immunocytochemistry, cells were cultured in slide chambers coated with poly-L-ornithine and fibronectin. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were then subjected to immunostaining procedures as described above. Anti-MAP2 and -anti-glial fibrillary acidic protein (GFAP) (ab929; abcam, Cambridge, United Kingdom) antibodies were used to detect neurons and astrocytes, respectively.
Generation of Jam-B homozygous mutant mice. The IRES ß-geo targeting vector (100 µg) was linearized with NotI and electroporated into 1 x 107 TT2 ESCs (45) as described previously (27). Electroporated ESCs were grown under G418 selection (300 µg/ml) for 1 week. Surviving ESC colonies were picked and expanded in 24-well tissue culture dishes. Once the cells became confluent, half of each clone was used for a cell stock, while the remaining half was further expanded. Genomic DNA was recovered from the expanded cells and used for Southern blot analysis using three probes (Fig. 3A). Two independently targeted ES cell clones were injected into eight-cell-stage embryos to generate chimeric mice. Chimeras were bred to generate F1 heterozygous animals, which were intercrossed to generate Jam-B homozygous mutant mice.
Genotyping of mice. Genomic DNA was extracted from tail tips of mice and was used for genotyping by Southern blotting or PCR. For Southern blot analyses, 10 µg of genomic DNA was digested with an appropriate enzyme (see Fig. 3B), electrophoresed on a 1% agarose gel, and transferred onto a nylon membrane. Membranes were hybridized with radiolabeled Neo, 5', or 3' genomic probes. For PCR, two sets of primers that amplify the deleted region of the Jam-B locus (550 bp) and a portion of the neomycin resistant gene (700 bp) were added together in each reaction. The sequences of these primers are as follows: Jam-B locus, 5'-CAGGTGCCTGAATTGATAGCTGCAGAACCC-3' and 5'-CAGCC AGAGC AGAAAGCTTGCTGATCAC-3'; neomycin resistant gene, 5'-GAACTGCAGGACG AGGCAGCGCGGC-3' and 5'-TATGAATTCCGAAGCCCAACCTTTCATAG-3'.
Northern blot analysis. Northern blot analyses were done as described previously (30) using poly(A)+ RNAs from wild-type and Jam-B mutant 12.5-dpc whole embryos. The filters were hybridized with either exon I-II, exon VI-X, or Neo probes. The exon I-II and exon VI-X probes encompass the sequence from –206 to +135 and that from +589 to +1103 of JAM-B mRNA, respectively, in which the adenine nucleotide of the translation initiation codon is +1.
Hematopoietic indices and FACS analysis of blood cells. Peripheral blood and bone marrow cells from wild-type and Jam-B mutant mice were recovered from the retroorbital plexus and the tibia/femur, respectively. Approximately 200 µl peripheral blood was mixed with 10 µl 100 mM EDTA (pH 7.5) to prevent coagulation, and half of each sample was applied to an automatic counter (Nihon Kohden, Tokyo, Japan) to determine the hematopoietic index. Mononuclear cells were recovered from the remaining blood samples by lysing erythrocytes with ammonium chloride and treating with 1 µl Fc blocker (BD Biosciences) at room temperature for 10 min. Subsequently, cells were incubated with biotinylated anti-Ly5.2, followed by fluorescein isothiocyanate-conjugated streptavidin. The cells were simultaneously stained with allophycocyanin (APC)-conjugated anti-Gr-1 and phycoerythrin (PE)-conjugated anti-Mac-1 or PE-conjugated anti-CD4, APC-conjugated anti-CD8, and PE-Cy7-conjugated anti-B220 antibodies. All antibodies were purchased from BD Biosciences. Multicolor analysis was performed using a FACS Vantage machine. Mononuclear cells were also prepared from bone marrow cells, and the number of CD34-negative KSL cells was quantitated as described by Osawa et al. (31).
Colony formation assay. Mononuclear cells from bone marrow were recovered as described above and overlaid with sodium metrizoate. Low-density cells were harvested, and lineage-positive cells were removed from these cells by utilizing biotinylated antilineage markers (Mac-1, Gr-1, B220, CD4, CD8, and TER119). These cells were applied to a FACS Vantage machine, and CD34-negative KSL cells were individually sorted to 96-well tissue culture dishes. These cells were cultured for 2 weeks with medium supplemented with stem cell factor (SCF), interleukin 3 (IL-3), thrombopoietin (TPO), and erythropoietin (EPO).
Histological staining and FACS analyses of testis.
Testes were recovered from 8-week-old wild-type and Jam-B mutant mice, fixed in Bouin's fix, and embedded in paraffin. Five-micrometer sections were prepared and were subjected to hematoxylin and eosin (H&E) staining. Staging of the seminiferous epithelium during spermatogenesis was determined as described by Russell et al. (35). For FACS analyses, testes were recovered from 8-week-old mice and treated with collagenase (1 mg/ml) for 15 min at 37°C. Subsequently, the samples were treated with a mixture of trypsin (0.2%) and DNase I (1.4 mg/ml) for 15 min at 37°C and then dissociated into a single-cell suspension with a pipette. Cells were incubated with anti-GFR1 (sc-6156; SANTA CRUZ) and biotin-conjugated anti-CD9 (clone KMC8; BD Bioscience) antibodies. These cells were further incubated with Alexa-488 Fluor dye-conjugated secondary antibodies from Invitrogen and APC-labeled avidin and then subjected to FACS analyses using a FACSCalibur device.
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RESULTS |
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Next, we investigated Jam-B expression in neural stem/progenitor cells. Primary neurospheres generated from 12.5-dpc mouse embryonic brains were dissociated and grown on dishes coated with poly-L-ornithine and fibronectin. The cells were cultured for an additional 48 h in the presence or absence of EGF and bFGF, which are required for preserving neural stem/progenitor identity (Fig. 2B). Like Nestin (14), Jam-B was strongly expressed in neurosphere cells cultured in the presence of EGF and bFGF, while markers of differentiation, such as Gfap and Map2, showed reduced expression compared to that observed in cells cultured without EGF and bFGF.
Next, we examined Jam-B expression in hematopoietic cells. HSC-enriched CD34-negative KSL cells (31), as well as a variety of other hematopoietic cell lineages, were isolated from either mouse bone marrow, thymus, or spleen by FACS, and RNA was prepared from sorted cells. Subsequently, reverse transcription-PCR was performed using a mixture of primer sets for Jam-B and ß-Actin mRNA. As shown in Fig. 2C, Jam-B expression is detected only in CD34-negative KSL cell populations. These data indicate that JAM-B is specifically expressed in HSCs but not in their differentiated derivatives, similar to the results seen for ESCs and neural stem/progenitor cells. However, the expression level of Jam-B in HSCs is likely not very high, since we were unable to detect Jam-B expression in a lineage-negative cell population from which CD34-negative KSL cells were derived.
Generation of Jam-B mutant ESCs. Next, we characterized the function of Jam-B by targeted disruption of the locus. First, we attempted to disrupt both alleles of Jam-B in ESCs to assess whether the gene is required for the maintenance of the stem cell state in ESCs. We prepared two targeting vectors, one of which carries a puromycin resistance cassette behind the PGK promoter and the other of which contains an IRES ß-geo (24) cassette (Fig. 3A). Both were designed to replace exons 3, 4, and 5 of Jam-B, which encode two immunoglobulin-like folds that are required for cell-cell contact (3, 8, 16, 19). We first introduced the puromycin targeting vector into E14tg2A ESCs by electroporation. PCR and Southern blot analysis revealed that the targeting vector correctly disrupted the Jam-B locus in 3 out of 75 independent puromycin-resistant clones. Subsequently, one of these three clones (Jam-BPuro/+) was used for subsequent targeting using the IRES ß-geo cassette. Among 21 G418-resistant colonies, we found that 17 were correctly targeted. This high frequency of recombination was likely due to the function of the IRES cassette in the vector, since it is assumed that the vector is able to support the reporter gene expression only in the case that the vector is integrated into one of the genes expressed in ESCs, including the JAM-B gene, but not into the genomic portions containing no functional gene or genes which are not expressed in ESCs (for details, see reference 24). In 13 clones, homologous recombination occurred in the previously targeted Jam-B allele, leaving the other allele intact (Jam-Bgeo/+), but in four cases, the vector disrupted the remaining wild-type allele (Jam-BPuro/geo) as desired (Fig. 4A).
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-fetoprotein (endoderm)-positive, cardiac troponin I (cardiac muscle)-positive, and MYOD (skeletal muscle)-positive cells were evident in teratomas from Jam-B mutant (Fig. 4H) and wild-type (data not shown) ESCs, and these cell populations were organized. From these results, we conclude that Jam-B is not required for preserving the stem cell state of ESCs. Jam-B knockout mice have no obvious abnormalities. Next, to elucidate the in vivo functions of Jam-B, we disrupted the Jam-B locus using the earlier-described IRES ß-geo targeting vector and used these ESCs to make chimeric mice. Germ line transmission of the targeted allele was obtained from two independent ESC clones. Southern blot analyses shown in Fig. 5B revealed that heterozygous intercrosses resulted in homozygous mutant mice in normal Mendelian ratios (Fig. 5A), with both male and female homozygous mutant mice being fertile with no apparent abnormalities (data not shown). Next, Northern blot analyses were performed using RNAs from wild-type and mutant 12.5-dpc whole embryos. The size of wild-type JAM-B mRNA is 2.9 kilobases, while the calculated size of mRNA from mutant Jam-B allele is 5.4 kilobases (exons I and II of the Jam-B gene plus IRES-ß-geo). As shown in Fig. 5C, we found that both expected mRNAs were visible with a probe bearing exon I and II (left panel). Moreover, transcripts from wild-type and Jam-B mutant alleles were also detected with exon VI-X (right panel) and Neo (middle panel) probes, respectively, and no unexpected band corresponding to an abnormal isoform of the JAM-B transcript was detected in any lanes of the three panels, validating our assumption that the Jam-B gene is indeed disrupted in homozygous mutant mice. However, we cannot completely eliminate the possibility that the transcript from the Jam-B mutant locus may express certain JAM-B functions, since the transcript contains a portion of the Jam-B coding sequence.
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JAM-B is not required in HSCs. Next, we examined the function of JAM-B in hematogenesis. First, we examined peripheral blood cell populations and saw no apparent difference between wild-type and Jam-B mutant mice in the numbers of white blood cells, red blood cells, and platelets. In addition, there were no differences in hemoglobin content and hematocrit levels (Table 1). Furthermore, FACS analysis with mononuclear cells from peripheral blood revealed that there was no significant difference between wild-type and mutant mice in myeloid and lymphoid cell populations (Fig. 7A). No differences were observed in CD34-negative KSL HSC cells recovered from bone marrow (Fig. 7B). Contents of CD34-negative KSL cells were 0.0043% and 0.0041% of total mononuclear cells from bone marrow of wild-type and Jam-B mutant mice, respectively.
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Normal testis morphology in JAM-B null mice.
As described above, both male and female Jam-B mutant mice were viable and fertile. However, it was surprising that Jam-B homozygous mutant males were fertile, since it is generally believed that JAM-B/JAM-C interaction in the testis plays a pivotal role in spermatogenesis (for details, see Discussion). Therefore, we performed histological analysis of the seminiferous tubules of 8-week-old wild-type and Jam-B mutant mice. As shown in Fig. 8A, we did not see any significant difference between wild-type and mutant mice at stage II-III (upper panels), stage V (lower panels), or any other stages (data not shown). We also quantitated the amount of spermatogonia cells. These cells are subdivided into A single, Apaired, and Aaligned spermatogonia according to their topographical arrangement on the basement membrane, and Asingle and Apaired cell populations in total can be identified by FACS as GFR1 and CD9 double-positive cells (for details, see reference 1). Our FACS analyses (Fig. 8B) revealed that again there was no prominent difference in the content of these spermatogonia cells between wild-type and Jam-B mutant mice. Thus, these results, together with the fertility of Jam-B mutant mice, indicate that spermatogenesis occurs normally in Jam-B mutant mice.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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However, although Jam-B is specifically expressed in ESCs, NSCs, and HSCs and not their differentiated derivatives, disruption of JAM-B function did not produce any overt abnormalities in any of these three types of stem cells. Indeed, we found that Jam-B mutant ESCs exhibit normal morphology and maintain pluripotency, generating teratomas containing cells from all three germ layers. We also generated Jam-B mutant mice and found that they are completely viable and fertile. Moreover, NSCs and HSCs recovered from Jam-B mutant mice are equivalent to those from wild-type mice in their stem cell properties.
The lack of fertility defects in Jam-B mutant mice is rather unexpected, based on data recently reported by Gliki et al. (11). The JAM-B protein is expressed in Sertoli cells, which assist spermatids undergoing cytodifferentiation into spermatozoa, while JAM-C, a protein whose amino acid sequence shows similarity to that of JAM-B (2-4, 9), is present in spermatids. These two proteins are known to mediate cell-cell contacts through heterophilic complex formation (2, 17, 18), and Gliki et al. (11) demonstrated a JAM-B-JAM-C interaction at the interface between spermatid and Sertoli cells. Moreover, they have shown that disruption of Jam-C function renders homozygous male mice completely infertile due to impairment of the morphological conversion of round spermatids into spermatozoa (11). Based on these data, it has been generally assumed that Jam-B knockout mice would exhibit similar spermatogenesis defects. However, our data demonstrate that a complete loss of Jam-B function does not affect fertility of either male or female mice. Histological analysis and quantitation of spermatogonia cells by FACS demonstrated that the seminiferous tubules appear normal in Jam-B mutant mice. These results indicate that disruption of JAM-B-JAM-C interactions between Sertoli and spermatid cells is not a major cause of the spermatogenesis defects observed in Jam-C mutant mice.
In summary, we found that disruption of Jam-B function does not cause any overt defects in three different types of stem cells or in spermatogenesis. However, since our experiments examined only embryonic and young-adult stages, it is possible that a phenotype exists in older animals. Although our data suggest that JAM-B function is not required for stem cell maintenance or spermatogenesis, it is possible that functional redundancy involving other JAM protein family members compensates for the loss of JAM-B. The three JAM protein family members, JAM-A, JAM-B, and JAM-C, have high amino acid sequence similarity (2-4, 9), and the expression profile of Jam-C in stem cells resembles that of Jam-B. Like Jam-B, Jam-C is expressed in NSCs and HSCs, with expression restricted to the multipotent state of these cells (13). No such difference in Jam-C expression was observed between pluripotent and differentiated ESCs, however. The variable penetrance of the lethal phenotype (60%) in Jam-C mutants (11) also suggests that JAM-B may compensate for loss of JAM-C. Thus, our future studies will examine ESCs, NSCs, and HSCs in JAM-B/JAM-C double-mutant or JAM-A/JAM-B/JAM-C triple-mutant mice.
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ACKNOWLEDGMENTS |
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This work was supported in part by the Ministry of Education, Science, Sports, and Culture, in particular by a Ministry Grant to Saitama Medical University Research Center for Genomic Medicine. This work was also performed as part of the Rational Evolutionary Design of Advanced Biomolecules (REDS) Project, Saitama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, supported by the Japan Science and Technology Agency, Japan.
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FOOTNOTES |
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