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1 Department of Medical Chemistry, Kansai Medical University, Moriguchi, 570-8506, Japan
2 Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Minato-Ku, Tokyo 108-8639, Japan
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Abstract |
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Introduction |
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Drosophila melanogaster ovo/shavenbaby (ovo/svb) gene encodes a zinc-finger protein, and the products of this gene act as transcription factors (Mevel-Ninio et al. 1991; Andrews et al. 2000) that are required for the differentiation of germ line cells and epidermis (Oliver et al. 1987; Payre et al. 1999). In mice, three genetically distinct homologues, Ovol1 (Dai et al. 1998), Ovol2 (Masu et al. 1998; also known as Movo, movo2) and Ovol3 (Li et al. 2002a) have been isolated as Drosophila ovo counterparts. Mouse OVOL1 was shown to be required for epidermal appendage differentiation (Li et al. 2002b); and mice lacking the Ovol1 gene display abnormalities in hair morphology, kidney development and male germ cell development (Dai et al. 1998; Li et al. 2005). Drosophila ovo and mouse Ovol1 have reported to act downstream of the Wnt-ß-catenin-LEF/T-cell factor signaling (Payre et al. 1999; Li et al. 2002b). On the other hand, the function of OVOL2 in mammals and its target gene(s) are thus far unknown. To address this question, we generated a functionally null mutant mice lacking Ovol2 exon 3, which encodes the first and second zinc-finger domains of four, by gene targeting. Herein we demonstrate that Ovol2–/– mice died during early embryogenesis due to defects in extraembryonic, embryonic angiogenesis and cardiac development, and suggest that Ovol2 plays a critical role in vascular angiogenesis during early embryogenesis.
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Results |
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In adult mice, Ovol2/Movo expression was earlier shown to be restricted to male germ cells of the testis (Masu et al. 1998). To clarify the role of OVOL2 in embryonic development, we analyzed its expression pattern during mouse development. Between embryonic days 8.5 and 10.5 (E8.5 and E10.5), Ovol2 expression was detected higher in the placenta than in the embryo, heart and yolk sac of wild-type mice by RT-PCR (Fig. 1A). The expression of Ovol2 mRNA was slightly detectable by Northern blot analysis in the whole conceptus at E7.5 (Fig. 1B). In the placenta, expression of the mRNA was detected at E8.5 by Northern blotting and maintained through mid-gestation (Fig. 1B). The size of the mRNA was the same as that of the one in the testis (Masu et al. 1998). In situ hybridization showed that Ovol2 mRNA was expressed in the chorionic plate at E8.5 (Fig. 1C,D) and later, in the labyrinthine layer of the placenta at E11.5 (Fig. 1F,G). Some of the giant trophoblast cells showed positive signals at E8.5 (Fig. 1C, arrows). Ovol2 was shown to be expressed in labyrinth trophoblast cells in a magnifying field (Fig. 1G). No signal was detected by sense probe (Fig. 1E,H).
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Since exon 3 encodes a portion of the DNA-binding zinc-finger domain of OVOL2 proteins, a targeting vector for removing exon 3 by Cre/loxP recombination was designed to study the function of OVOL2 proteins in vivo (Fig. 2A). The deletion of exon 3 leads to a frame-shift mutation from exon 2 to exon 4. After homologous recombination and Cre transfection, the resistant E14.1 ES cell clones were confirmed for recombination by Southern hybridization (Fig. 2B). Two independent ES cell clones were used to generate chimeric mice, and the mutation was transmitted through the germ line. Offsprings derived from both clones showed the same phenotype. Heterozygous mice of both sexes were healthy and fertile, and no overt abnormal phenotype was observed. Among 266 live-born progeny from heterozygous intercrosses, 80 wild-type and 181 heterozygous offspring were recovered; however, no homozygous mutant offspring was found (Table 1). To determine the time of embryonic death and to characterize the morphological phenotype of the mutant embryos, we recovered the embryos at different stages of gestation and analyzed their genotypes by PCR (Fig. 2C). No band was detected by RT-PCR analysis for detection of targeted exon 3 from the mutant placenta (Fig. 2D). From E7.5 to E12.5, Ovol2–/– embryos could be detected with almost the expected Mendelian frequency (Table 1). At E8.5, the Ovol2–/–embryo was grossly normal but showed a sign of the cardiac defect, that is, a small heart (Fig. 2E, arrows). The difference in embryonic development between wild-type and Ovol2–/–embryos became more evident at E9.5 (Fig. 2F,G). Wild-type embryos finished turning and showed a well-developed heart, three branchial arches and a head region. Their hearts beat strongly, and the blood cells were easily detected in the heart and vessels, suggesting the initiation of the blood circulation. In contrast, Ovol2–/–embryos were small in size and abnormal in structure, such as having a very small heart, open head folds and only the first branchial arch (Fig. 2F). Their small hearts contracted irregularly, but there were few blood cells in the heart and vessels. Evidence of hemorrhages was observed in both capital and caudal regions (Fig. 2F, arrows). The abnormality was also observed in the extraembryonic tissue; that is, the yolk sac failed to develop large vitelline vessels (Fig. 2G). Thus, mutant embryos lacking the Ovol2 gene died in utero between E9.5 and E10.5 of gestation, demonstrating that OVOL2 was essential for embryonic development.
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To elucidate the cause of the poor blood circulation observed in Ovol2–/– embryos at E9.5 (Fig. 2), we analyzed the development of the vascular system by immunostaining for PECAM-1, a specific marker for endothelial cells. Histological analysis showed that E8.5 Ovol2–/– embryos exhibited the normal structure of major organ systems such as the neural tube and foregut, and of the dorsal aortas, just as did the wild-type embryos (Fig. 3A). In PECAM-1-immunostained Ovol2–/– embryo at E8.5, the dorsal aortas were formed as well as they were in wild-type embryos, whereas the vascular formation in the head region and heart endocardium remained undeveloped (Fig. 3B,C). The defects in the heart and vascular formation were more evident in E9.5 embryos (Fig. 3D). In wild-type embryo, branched vessels were seen in the head; and intersomitic vessels, formed by angiogenesis, were noted, as was heavy staining in the ventricle chamber. In Ovol2–/– embryos, blood vessels were disorganized throughout the whole body (Fig. 3D). There was a small tissue clump in the heart, and a few disconnected vessels in the head. Typical intersomitic vessels, which were arranged in segments and located between somitic boundaries in the wild-type embryo, were absent; and an excessive number of disorganized vessels were found in the caudal region (Fig. 3D, arrowheads). These results demonstrate that Ovol2–/– embryos had serious defects in heart development and angiogenesis, but not in vasculogenesis.
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In normal embryos at E8.5, the heart tube consisted of myocardial and endocardial layers; and looping had been initiated (Fig. 3B,H). In mutant embryos, the heart tube was also formed and these two layers were observed (Fig. 3H), but the tube had not initiated looping (Fig. 3C). Moreover, the endocardium and aortic sacs were barely observable (Fig. 3C). Histological analysis of E9.5 wild-type hearts showed normal chamber formation and trabeculation in the ventricle chamber (Fig. 3I,J). In contrast, Ovol2–/– embryos showed ventricular chamber formation, as evidenced by the expression of the ventricular myosin light chain gene (Mlc2v, data not shown), but a reduced amount of endocardial tissue and abnormal chamber formation, as well as a lack of myocardial trabeculation (Fig. 3I,J). These results demonstrate that Ovol2–/– embryos had serious defects in the growth of myocardial and endocardial layers, resulting in the abnormal looping and chamber formation.
Placental abnormalities of Ovol2–/– embryos
The fusion of allantois to chorion, the first step of placental development, was unaffected in Ovol2–/– embryos at E8.5–E9.0 (data not shown). At E9.5, the allantoic vessels in the wild-type placenta extended to the labyrinth layer, where extensive intermingling occurred between maternal blood sinuses and fetal blood vessels (Fig. 4A). Blood vessels were also shown by the detection of Flk1, a VEGF receptor expressed in endothelial cells (Fig. 4A,E). The invasion of the allantoic vessels and the number of fetal blood cells were slightly less in the Ovol2–/– placenta than in the wild-type placenta (Fig. 4E), but there was no difference in the placental cell proliferation and cell viability between them (data not shown). We next compared the expression of several cell markers between Ovol2–/– and normal placentas. All of three trophoblast cell markers, placental lactogen-1 (PL1; giant cell layer), 4311 (spongiotrophoblast layer) and Gcm1 (syncytiotrophoblast in the labyrinth layer) were normally expressed in Ovol2–/– placenta at E9.5 (Fig. 4B–D). In the wild-type placenta at E10.5, the fetal vessels branched and extensively invaded the labyrinth layer; and this layer was itself expanding (Fig. 4F). In the Ovol2–/– placenta, however, this invasion process was retracted; and only a few fetal blood cells were present in the chorion (Fig. 4F). Giant cell and spongiotrophoblast layers appeared normal in Ovol2–/– placenta (data not shown). The branched pattern of Gcm1 expression was not detected and not expanded in the labyrinth layer in Ovol2–/– placenta (Fig. 4G,H). Furthermore, the Flk1 expression was not expanded in the labyrinth layer in Ovol2–/– placenta as compared with that of the Ovol2+/– placenta (Fig. 4I). These results demonstrate that Ovol2 was not required for the initial establishment of allantoic vessels and differentiation of trophoblast cells, but was needed for the support of normal branching or elongation of the vessels and labyrinthine layer in the placenta.
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The common phenotype appeared in mutant embryo and yolk sac was assumed that the endothelial cell is one of the target cells in Ovol2 mutant. We isolated mouse endothelial cells from embryos at E11.5 by MACS cell separation system using anti-PECAM-1 antibody. Ovol2 expression was detected by RT-PCR in both endothelial cells (EC) and PECAM-1 negative embryonic cells (Others), but the expression levels in three endothelial cell lines, that is, MS1, bEND.3 and C166, were higher than that in hematopoietic cell lines F4N and BW5147 (Fig. 5A). To confirm the function of OVOL2 in endothelial cells, we used the siRNA approach to knockdown Ovol2 expression in endothelial cell line MS1. Because of the low expression level of Ovol2, we evaluated the siRNA efficacy by the reduction in the ß-galactosidase (ß-gal) activity of OVOL2-ß-gal fusion protein in co-transfected MS1. As shown in Fig. 5B, Ovol2 siRNAs 242 and 590 blocked the OVOL2-ß-gal fusion protein activity by 80%–90% but not the ß-gal activity. The negative control siRNA failed to modify the fusion protein activity. As shown in Fig. 5C, the capillary-like network formation on Matrigel was inhibited in the cells transfected with the Ovol2 siRNAs 242 and 590, but not in those cells treated with the negative control siRNA. When the inhibition of the capillary-like network formation was assessed in terms of total tube length, the siRNA 242 and 590 inhibited it by 70% and 40%, respectively (Fig. 5D). In contrast, Ovol2 knockdown by siRNA showed no effects on the cell proliferation (Fig. 5E) and cell viability (Fig. 5F). These results suggest that OVOL2 was essential for the angiogenesis in endothelial cells.
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To identify an OVOL2 target gene(s) causing the defects in Ovol2 mutants, we analyzed the expression levels of genes known to be critically involved in vascular formation and heart development by RT-PCR. All genes examined were expressed in Ovol2–/– embryos, indicating that OVOL2 is not absolutely required for their expression (Supplementary Fig. S1). Endothelial cell growth factors, VEGF and basic fibroblast growth factor (bFGF) were moderately increased, but VEGF receptors, Flt1 and Flk1, and angiogenic factors and their receptors examined here appeared no difference in the embryo, yolk sac and placenta between wild-type and Ovol2–/– mice at E9.5 (Supplementary Fig. S1B) as well as at E8.5 (Supplementary Fig. S1A). The expression of 
-smooth muscle actin (SMA) was also similar to that in wild-type embryo. Furthermore we examined the expression of genes related to heart morphogenesis, that is, GATA4, Nkx2.5, Hand1, Hand2 and MEF2C, and of those involved in the development of the ventricle endocardium and trabeculation, that is, Flk1, Flt1, Flt4, Ang1, Tie2, vascular endothelial-cadherin (VE-Cadherin) and VEGF by RT-PCR. All genes examined showed similar levels of the expression in wild-type and Ovol2–/– hearts at E8.5 and E9.5 (Supplementary Fig. S1).
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Discussion |
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The cardiovascular system is the first system to appear in the developing embryo, and its establishment and functioning are critical for fetal survival beyond E9.5. In Ovol2–/– embryos, the endothelial cells were observed (Fig. 3) and normally expressed the specific cell markers (Flk1, Flt1, Tie2 and VE-cadherin, Supplementary Fig. S1). The hematopoietic stem cells showed the normal differentiation potency (Table 2). The primary vascular plexus in their yolk sac and dorsal aorta and heart endocardium were present at E8.5 (Fig. 3). However, the large vitelline vessel and vessel branching in the yolk sac were completely defective at E9.5 (Fig 3). In the mutant embryo, the vascular formation in the head region was still undeveloped at E8.5, and the intersomitic vessels were disorganized at E9.5 (Fig. 3). By repressing the OVOL2 expression, we demonstrated the endothelial cells to have a reduced ability for tube formation (Fig. 5). In embryos, the endocardium and dorsal aortas are generated by vasculogenesis; and brain, kidney and intersomitic vessels, by angiogenesis (Drake & Fleming 2000). Thus, our results demonstrate that the vasculogenesis and hematopoiesis occurred normally in the Ovol2–/– embryo but that angiogenesis was blocked in both extraembryonic and embryonic vessels by the abnormality in endothelial cells.
In the E8.5 Ovol2–/– embryo, the heart tube was formed, consisting of an outer myocardium, and a reduced endocardium; but no myocardial trabeculation was found. In addition to the abnormal chamber formation, the aortic sac was undeveloped and the pharyngeal region was aberrant (Fig. 3). Defective looping is one of the typical phenotypes reported in many types of gene-deficient mice, for example, those deficient in Nkx2.5 (Tanaka et al. 1999), Mef2c (Lin et al. 1997), Hand1 (Firulli et al. 1998) or Hand2 (Srivastava et al. 1997), but these genes were normally expressed in the Ovol2–/– embryo (Supplementary Fig. S1), indicating that OVOL2 regulates the heart formation through another signaling system.
The chorioallantoic fusion occurred normally, and the allantoic vessels started to invade the chorionic plate of the Ovol2–/– placenta at E9.5. But the branching and expansion of the allantoic vessels and labyrinthine layers were retarded at E10.5 (Fig. 4). Consistent with our observations on the embryo and the yolk sac, these results support the findings that vessel branching and further invasion by angiogenesis were prevented in the Ovol2 mutant. The poor circulation in the Ovol2–/– embryo may have caused these defects in the placenta after 1 day, but the highly Ovol2 mRNA expression in the labyrinthine layer suggests a unique role for Ovol2 in placental development.
To identify an Ovol2 target gene, we analyzed the expression levels of genes known to be critically involved in vascular and heart development (Supplementary Fig. S1). Most genes were not significantly different between wild-type and Ovol2–/– embryos except for several genes. The angiogenic growth factors, VEGF and bFGF, were up-regulated in Ovol2–/– embryos at E9.5 (Supplementary Fig. S1). VEGF and bFGF are known to directly implicate in the endothelial cell proliferation and migration, and be induced the expression by the hypoxic condition. It suggests that the global impairment of blood supply and circulation in Ovol2–/– embryos is expected to lead to embryonic hypoxia, enhancing the production of these angiogenic factors (Kuwabara et al. 1995). Recent studies have revealed that several signaling pathways are important during vasculogenesis and angiogenesis: VEGF, TGFß, Ang1, ephrin and Notch signaling (Dickson et al. 1995; Krebs et al. 2000; Yancopoulos et al. 2000; Uyttendaele et al. 2001). VEGF signal is essential for the blood vessel formation by both vasculogenesis and angiogenesis, and heart formation (Carmeliet et al. 1996). In the mutant mice for VEGF receptors, Flt-4, Neuropilin-1/-2 and VEGF-A, showed somewhat similar phenotypes of the defects in embryonic and extraembryonic vascular formation and/or heart development (Carmeliet et al. 1996; Dumont et al. 1998; Takashima et al. 2002). However, since these gene expressions of Ovol2–/– mice were not altered (Supplementary Fig. S1), these genes might not be regulated by Ovol2 directly. Reciprocal interactions between vascular endothelial cells and mesenchymal cells are essential for angiogenesis and maintenance of the vasculature. TGFß, Ang-1/Tie2, ephrinB2/EphB4 and Dll4/Notch1, four signaling are essential to angiogenesis (Dickson et al. 1995; Sato et al. 1995; Adams et al. 1999; Krebs et al. 2000; Uyttendaele et al. 2001). Although no difference was detected in the expression of the genes related to these signals (Supplementary Fig. S1 and data not shown), the mutant phenotypes of these related genes are similar to that of Ovol2, and this suggested that Ovol2 may regulate the expression of the gene(s) in peri-endothelial cells to induce angiogenesis.
Similar to the endothelial–mesenchymal interaction in angiogenesis, reciprocal interactions between the endocardium and myocardium play an essential role in cardiogenesis by the involvement of genes common to angiogenesis such as Ang1/Tie2 and VEGF (Sato et al. 1995; Carmeliet et al. 1996). Nkx2.5, GATA4, Hand1/Hand2 and MEF2C are essential for cardiogenesis (Srivastava et al. 1995; Kuo et al. 1997; Lin et al. 1997; Tanaka et al. 1999). All genes examined showed similar levels of the expression in wild-type and Ovol2–/– hearts at E9.5 (Supplementary Fig. S1), suggesting that down-regulation of these cardiac regulatory genes may not be involved in the cardiac defects in the Ovol2 mutant.
Recently, mouse Ovol1 has reported to regulate the expression of Id2 gene (Li et al. 2005). The Id proteins are a family of four related proteins, and Id1 and Id3 are required for the angiogenesis during mouse brain development and tumorigenesis (Lyden et al. 1999). We also analyzed the expression of these Id genes, but did not detect the difference between wild-type and mutant embryos (Supplementary Fig. S1).
In conclusion, our results indicate that Ovol2 function is required for the endothelial cell growth during heart development and angiogenesis of extraembryonic and embryonic vessels. The target gene of Ovol2 is unknown, but it may be expressed in endothelial cells and function in both extraembryonic and embryonic tissues.
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Experimental procedures |
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Ovol2/Movo gene (DDBJ accession nos. AB101297, AB101298 and AB101299) was isolated from a 129/SvJ genomic library, and the targeting vector was generated from a 10-kb EcoRI-SacI fragment around Ovol2 exon 3. A single FRT site (Nakano et al. 2001) was ligated to each end of the PGKneobpA cassette (Soriano et al. 1991), and this FRT-Neo cassette was inserted into the PstI site downstream of the Ovol2 exon 3. A loxP site was inserted into the ApaLI site upstream of Ovol2 exon 3 and at the PstI site at which the FRT-Neo cassette had been inserted. A DT-A cassette (Yagi et al. 1993) was ligated to the 5' end of the targeting vector. The linearized targeting vector was electroporated into E14.1 ES cells, and selection was done with G418. The resistant clones were screened for homologous recombination by Southern blot analysis. To delete the Ovol2 exon 3, we infected the targeted ES cells with a recombinant adenovirus that expresses Cre recombinase transiently (Kanegae et al. 1995). The Cre-mediated recombinants were screened by PCR and confirmed by Southern blot analysis. Chimeric mice were generated from two ES cell clones, 3D2 and 4D3, by the aggregation of C57BL/6J x BDF1 8-cells. Male chimeras were mated with C57BL/6J females, and the heterozygotes were intercrossed. Genomic DNAs were isolated from ES cells, tails, and embryonic tissues and then digested with restriction enzymes (PvuII, HindIII + XhoI), and Southern blot analysis was performed by using a 5' end probe and a 3' end probe. The sizes of HindIII-XhoI fragments were 7.5 kb (wild-type allele) and 6 kb (targeted and mutant alleles); and those of PvuII fragments, 8.8 kb (wild-type allele), 8.3 kb (mutant allele) and 4.4 kb (targeted allele). For PCR analysis, specific bands were amplified by using the primers 5'-CATAGCCCATGTGTGGCTGCTG-3' and 5'-GCCGGCCTTAAAACATCCCAC-3'.
Northern blot analysis and RT-PCR
Northern blot analysis was performed with 10 µg of total RNA by using full-length Ovol2 cDNA (Movo-A) and GAPDH cDNA as probes. For RT-PCR analysis, total RNAs were treated with DNase I and then reverse transcribed with p(dN)6 random primer. The sequences of RT-PCR primers are shown in the Supplementary Table S1.
Histochemistry and in situ hybridization
Embryos and tissues were fixed with 4% paraformaldehyde/PBS at 4 °C overnight, paraffin-embedded, sectioned at 3–5 µM, and stained with hematoxylin–eosin (HE). Whole-mount immunostaining was performed by using anti-PECAM-1 (CD31) antibody (MEC13.3, BD Biosciences, Bedford, MA) as described previously (Schlaeger et al. 1995). For in situ analysis, fixed samples were frozen, sectioned at 10–15 µM with a cryostat, and hybridized with digoxigenin-labeled cRNA probes according to the manufacturer's protocol (Roche Diagnostic, Mannheim, Germany). The DNA fragments of PL1, 4311, Gcm1 and Flk1 were amplified from murine placental cDNA by PCR using the primers shown in the Supplementary Table S1. cRNA probes were synthesized from these DNA fragments and full-length Ovol2 cDNA (Movo-A) by use of a digoxigenin labeling system (Roche Diagnostic).
Isolation of endothelial cells from mouse embryos
Mouse endothelial cells were isolated from E11.5 embryos by the MACS cell separation system (Miltenyi Biotec, Bergisch Gladbach, Germany) using anti-PECAM-1 antibody (MEC13.3) and anti-Rat IgG microbeads (Miltenyi Biotec) without trypsinization as described previously (Marelli-Berg et al. 2000). The flow-through cells and eluted endothelial cells were cultured as described previously (Marelli-Berg et al. 2000). The purity of endothelial cells was 80% determined by anti-PECAM-1 immunostaining. In flow-through cells, no cell was stained by anti-PECAM-1 immunostaining.
Transfection assay
Mouse cell lines, MS1 (pancreatic endothelial cell), bEND.3 (brain endothelial cell line), C166 (yolk sac endothelium), F4N (erythroleukemia cell) and BW5147 (T cell) were obtained from ATCC, ATCC, ECACC and HSRBB, respectively, and grown according to supplier's protocol. ß-Gal and luciferase expression plasmid, pEF-LacZ and pEF-Luc, were constructed to express them under an EF promoter (Unezaki et al. 2004). For the expression of an OVOL2- ß-gal fusion protein, the MOVO-B coding region was inserted at the upstream of ß-gal (pEF-MOVO-LacZ). Specific siRNAs directed against Ovol2 were designed by using siDirect (Naito et al. 2004). The sense-strand sequences were the following: 242, 5'-CUCUGUGAUUCACAACUGU-3'; 590, 5'-GCAUGUGAACAGUGACCAU-3'. The 21-mer both-strand siRNAs were synthesized and annealed to form a 3' overhang of their own RNA sequence by RNAi (Tokyo, Japan). A negative control siRNA, having 5'-AUCCGCGCGAUAGUACGUA-3' as its sense strand, was purchased from B-Bridge International (Sunnyvale, CA). The efficacy of the siRNA was examined by the ability to reduce the signals from the reporter-target construct, pEF-MOVO-LacZ (Kumar et al. 2003). The siRNAs and plasmid DNAs were transfected using Lipofectamine 2000 and Lipofectamine (Invitrogen, Carlsbad, CA), respectively, according to the manufacturer's protocols. Briefly, MS1 cells in 24-well plates (3 x 104 cells/well) were transfected with 10 pmol of siRNAs and 1.6 µg of plasmid DNA (pEF-LacZ or pEF-MOVO-LacZ) and 10 ng of pEF-Luc as an internal control. After 24 h, cell lysates were harvested and ß-gal and luciferase activities were assayed as described previously (Unezaki et al. 2004). For cell proliferation assay, cells were incubated with bromodeoxyuridine (BrdU) last 2 or 4 h at a final concentration of 10 µM. After 24 h of transfection, the cells were fixed and treated with 2N HCl and 0.5% Triton-X100, and the BrdU incorporation was detected by anti-BrdU antibody (BU33, Sigma, St. Louis, MI), followed by the LSAB/HRP reagent (Dako, Kyoto, Japan). The numbers of total and BrdU-positive cells were counted by the IMAGE J software <http://rsb.info.nih.gov/ij/>. Cell viability was measured by Trypan blue exclusion assay 24 h after transfection. Three to five independent transfection experiments were carried out and significance of differences was calculated by using Student's t-test.
Microtubule formation assay
MS1 cells were plated at 1–1.2 x 105 cells and 50 pmol of siRNAs were introduced into them by use of Lipofectamine 2000. After 24 h, the cells were trypsinized and counted, and 7 x 104 cells were plated onto 48-well plate coated with 150 µL of Matrigel (BD Bioscience). The cells were observed by microscopically, and total tube length was determined. Significance of differences was calculated by using Student's t-test.
In vitro colony-forming assay for yolk sac cells
In vitro colony-forming assay was carried out as described previously (Sinclair et al. 2002). Briefly, yolk sacs from E9.5 embryo were separated and mechanically dispersed in Iscove's modified Dulbecco's medium. The cells were counted and plated in Methocult GF-M3434, a cytokine-supplemented methylcellulose medium (StemCell Technologies, Vancouver, Canada). After an 8-day culture period, the number of colonies was scored. Significance of differences was calculated by using Student's t-test.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: ito{at}takii.kmu.ac.jp
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References |
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Accepted: 5 March 2007
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