Expression and Localization of l-Afadin during Early Embryogenesis
We first examined expression and localization of l-afadin during mouse early embryogenesis. At embryonic day 6.5 (E6.5), embryos developed to egg cylinders containing embryonic and extraembryonic regions and proamniotic cavities. The embryonic ectoderm was composed of high columnar epithelial cells surrounded by the visceral endoderm. Yolk sac and ectoplacental cone were clearly observed in this stage. Immunofluorescence microscopy of E6.5 embryos revealed that l-afadin was localized at the most apical regions of cell–cell adhesion sites, called the junctional complex regions, of the entire embryonic ectoderm, whereas the signals of F-actin were observed along the entire cell surface ( Aa and Ab). l-Afadin was hardly detected in the extraembryonic regions such as the visceral endoderm. At E7.0, embryos underwent primitive streak formation and mesoderm generation. Gastrulation began by the recruitment of embryonic ectodermal cells to the primitive streak, followed by exfoliation of cells from the primitive streak. Whole-mount immunohistochemistry revealed marked expression of l-afadin in the primitive streak and the migrating paraxial mesoderm ( B). At E7.5, l-afadin was highly concentrated at the junctional complex regions in the primitive streak region (neuroepithelium) and the neural fold/groove region, but it was hardly detected in other areas of the ectoderm (, Ca–Ce). It remains to be clarified whether l-afadin is downregulated or whether the proportion of ectodermal cells with low expression of l-afadin increases. By E8.5, normal embryos completed gastrulation and began organogenesis. The primitive streak regressed and newly organized tissues developed. High expression of l-afadin was detected in the tail bud, somites, and the paraxial mesoderm, which is being reorganized to form somites, neural tube, and intraembryonic coelomic cavity/pericardio-peritoneal canal that gives rise to pleura and pericardium ( Da). l-Afadin was highly concentrated at the junctional complex regions in neural tube, somites, and pericardio-peritoneal canal (, Db–Df). These results indicate that l-afadin is highly expressed in a restricted set of epithelial structures and highly concentrated at their junctional complex regions.
Figure 1 Expression and localization of l-afadin during early embryogenesis. Embryos at various stages were subjected to immunofluorescence microscopy and/or whole-mount immunohistochemistry. (A) Immunofluorescence microscopy of E6.5 embryos (transverse section). (more ...)
Targeting of the Afadin Locus
To determine the function of l-afadin in these epithelial structures, the mouse afadin gene was disrupted by homologous recombination. A targeting vector was designed so as to delete the exon 2 ( A). The linearized targeting vector was introduced into ES cells and subjected to selection using G418. To screen for homologous recombination events, genomic DNAs from G418-resistant clones were subjected to Southern blot analysis with a 5′ probe. The wild-type afadin allele displayed a 13.9-kb band on Southern blotting of HindIII-digested DNA, whereas the disrupted locus showed a 5.0-kb band ( B). Corrected targeting was confirmed by Southern blot analysis with a 3′ probe. The wild-type afadin allele displayed a 13.9-kb band, whereas the disrupted locus showed a 7.8-kb band. Three different ES clones (A46, A59, and A97) with the targeted allele were separately injected into host blastocysts, and the blastocysts were transferred to the uteri of pseudopregnant female mice. Germline transmission of the targeted allele was achieved with all ES clones. Inheritance of the targeted allele was determined by Southern blot analysis of the genomic DNA isolated from tail biopsies ( C). The heterozygous (afadin+/−) mice appeared normal compared with the wild-type littermates. The afadin+/− mice were intercrossed and genotypes of the progeny were determined by Southern blot or PCR analysis using tail DNAs ( C). No homozygous (afadin−/−) mice were detected among 82 progeny analyzed. These results indicate that deficiency of afadin causes embryonic lethality.
Developmental Defects of Afadin−/− Mice during Early Embryogenesis
Embryos were isolated at various stages of gestation and their genotypes were determined (). Distribution of each genotype examined at E7.5–E9.5 followed the Mendelian law, whereas no homozygous embryos were detected from E10.5. At E6.5, gross morphological analysis did not distinguish the homozygous embryos from the wild-type and heterozygous littermates (data not shown), indicating that implantation and egg cylinder formation occur normally in the absence of afadin. To examine the presence of residual maternal afadin, which may affect implantation and egg cylinder formation in the homozygous embryos, preimplantation embryos at E3.5 (early blastocysts) were cultured and their levels of l-afadin were determined by immunofluorescence microscopy. Of 20 embryos examined, 16 showed weak but significant staining, whereas the remaining did not show any signal (data not shown). It is most likely that the latter embryos are homozygous and that there is no residual maternal afadin in the homozygous embryos. In contrast to embryos at E6.5, it was easy to distinguish the homozygous embryos from the wild-type and heterozygous littermates in embryos at E7.5–E9.5. Compared with wild-type embryos, the architecture of the homozygous embryos was apparently distorted and reduced in size (, Aa–Ac). Of note, however, is that their extraembryonic regions, including ectoplacental cone and yolk sac, developed normally, indicating that the anomalies are basically restricted to embryos proper. Whereas the anterior–posterior distinction could easily be made by gross appearance, and some vascular system and blood were detectable, the homozygous embryos were always flat and short, and no landmark tissues such as heart developed at this stage. The anomalies specific for these embryos proper in afadin−/− embryos are consistent with the expression patterns of l-afadin.
Genotypes of Progeny from Intercrosses of Afadin Heterozygous Mice
Figure 3 Developmental defects of afadin−/− embryos during early embryogenesis. (A) Gross appearance. (Aa) E7.5 embryos; (Ab) E8.5 embryos; and (Ac) E9.5 embryos: left, wild-type embryos; and right, afadin−/− (more ...)
Disorganized Ectoderm in Afadin−/− Embryos during Gastrulation
In wild-type embryos at E7.5, delamination of mesodermal cells from the primitive streak was undertaken in a strictly polarized manner, thereby recruiting mesodermal cells into the space between the embryonic ectoderm and the visceral endoderm ( Ba). Of importance is that the integrity of epithelial structures of the ectoderm, including the primitive streak and neural groove, is maintained even under the stimulation to induce delamination. In afadin−/− embryos at E7.5, generation of mesodermal cells at this space did occur, indicating that mesoderm induction itself occurs normally (, Bb–Be). However, these mutant embryos had the wider space between the ectoderm and the endoderm, where more mesodermal cells were observed compared with wild-type embryos. Ectodermal cells of afadin−/− embryos appeared flat or cuboid, and their polarized epithelial structures appeared to be entirely impaired (, Bb–Be). At the region corresponding to neural fold/groove (from the anterior region to the distal region), the ectoderm became multilayered and appeared as a cell mass (, Bb–Be). At the posterior region corresponding to the primitive streak, the ectoderm was invaginated toward the amniotic side (, Bc and Be; see also Bc, and , Ca, Cc, Ga, and Gc). The invaginated ectoderm often ran along with the ectoderm of the lateral region, resulting in the appearance of two layers. The space between the two layers corresponded to the amniotic cavity, which was compressed to an inverted U-shape. Cells were detected at the space surrounded by the invaginated ectoderm. Furthermore, in afadin−/− embryos, amniotic and exocoelomic cavities did not develop normally and formation of allantois was not observed ( Be). In contrast to the severe defects of the embryonic ectoderm, a single-layered epithelial structure in the endoderm remained intact (, Bb–Be). Hence, all of these phenotypes may be an outcome of abnormal progression of amniotic and chorionic membrane formation from the most proximal region of the primitive streak. In wild-type embryos at E8.5, tissues with epithelial structures, such as neural groove, intraembryonic coelomic cavity, and somites, were clearly evident ( Ca). In afadin−/− embryos, consistent with histological findings showing generation of mesodermal cells, gross appearance showed that somite-like blocks and some vascular structures were detected (data not shown), although no epithelial structure of somites was established ( Cb). Formation of neither neural tube nor heart was observed.
Figure 4 Abnormal primitive streak in afadin−/− embryos. E7.5 embryos were subjected to whole-mount in situ hybridization with the digoxigenin-labeled antisense RNA of T as a probe, sectioned, and counterstained with neutral red. (A) Wild-type (more ...)
Figure 5 Disorganized ectoderm in afadin−/− embryos. Transverse sections of E7.5 embryos were doubly stained with the anti–E-cadherin antibody and the anti-PDGFRα or anti-Flk1 antibody for immunofluorescence microscopy. (A and E) (more ...)
To further dissect the developmental defects of afadin−/−
embryos, we next investigated expression of E-cadherin and mesoderm markers, including T
, PDGFRα, and Flk1 at E7.5. Consistent with an earlier observation (Wilkinson et al. 1990
was highly expressed in the primitive streak and its nascent mesoderm in wild-type embryos (, Aa and Ab). In afadin−/−
embryos, the T
-positive area appeared to be divided into two portions that corresponded to the most posterior regions of the two-layered ectoderm (, Ba–Bd). At the primitive streak stage, E-cadherin is expressed in the entire embryonic ectoderm (Takeichi 1988
). PDGFRα is expressed in the paraxial mesoderm (Takakura et al. 1997
), and Flk1 is expressed in the proximal lateral mesoderm and the extraembryonic mesoderm (Kataoka et al. 1997
). After completion of exfoliation from the primitive streak, E-cadherin was downregulated, and PDGFRα and Flk1 were expressed in the mesodermal cells (, Aa–Ac and Ea–Ec). In afadin−/−
embryos, E-cadherin–negative and PDGFRα-positive cells were detected at the space between the ectoderm and the endoderm (, Ba–Bc, Ca–Cc, and Da–Dc). These cells corresponded to the mesodermal cells of the paraxial region. At the proximal region of the primitive streak, E-cadherin–positive cells were jammed at the space between the ectoderm and the endoderm (, Ba, Bc, Fa, and Fc; see also , Ca, Cc, Da, and Dc). The staining for E-cadherin clearly demonstrated that the E-cadherin–positive ectoderm was invaginated from the posterior region toward the amniotic side (, Ca and Cc). The most posterior region of the two-layered ectoderm corresponded to the area positive for T
(, Ca and Cc; see also Bc). E-cadherin–negative and PDGFRα-positive cells were detected at the space surrounded by the invaginated ectoderm. These cells appeared to migrate from the primitive streak. At the regions corresponding to neural fold/groove (from the anterior region to the distal region), the multilayered cells were E-cadherin–positive (, Ba, Bc, Ca, Cc, Da, and Dc). At the distal region, some cells in the cell mass expressed not only E-cadherin but also PDGFRα (, Da–Dc). This cell mass was surrounded by a layer of the ectodermal cells that were E-cadherin–positive and PDGFRα-negative. Similar observations were obtained with the double staining for E-cadherin and Flk1, except that Flk1 was not expressed in the cell mass (, E–H). These observations strongly suggest that the major histological basis of the developmental defects of afadin−/−
mice is disorganization of the embryonic ectoderm, and that distorted placement of various cell lineages is the secondary outcome of this disorganization.
Figure 6 Disorganized cell–cell junctions of the embryonic ectoderm in afadin−/− embryos. Transverse sections of E7.5 embryos were doubly stained with the anti–E-cadherin and anti–ZO-1 antibodies. (A and B) Wild-type embryos; (more ...)
Disorganized Cell–Cell Junctions of the Ectoderm in Afadin−/− Embryos
To investigate whether or not the apparatus for maintaining cell polarity is disturbed in the embryonic ectoderm, we examined the localization of E-cadherin and ZO-1 in afadin−/− embryos at E7.5. At the primitive streak region (neuroepithelium) and the neural fold/groove region in wild-type embryos, E-cadherin was concentrated at the junctional complex regions, although its signals were detected along the lateral membrane (, Aa, Ac, Ba and Bc). ZO-1 was exclusively localized at the junctional complex regions of the embryonic ectoderm (, Ab, Ac, Bb, and Bc). At the primitive streak region in afadin−/− embryos, E-cadherin hardly showed such an organized concentration as observed in wild-type embryos (, Ca, Cc, Da, and Dc). In the cell mass from the anterior region to the distal region, E-cadherin was distributed diffusely over the entire cell surface (, Ca, Cc, Ea, Ec, Fa, and Fc). The localization of ZO-1 was also disturbed in afadin−/− embryos. At the primitive streak region, ZO-1 was mainly localized at the most apical regions, but the signals were also detected in the basal regions (, Cb, Cc, Db, and Dc). In the cell mass, ZO-1 showed dotty signals in a random manner (, Eb, Ec, Fb, and Fc). These results indicate that deficiency of afadin induces disorganization of cell–cell junctions of the embryonic ectoderm.
Demonstration of Ectoderm-specific Afadin Function in an In Vitro Model System
The results of afadin−/− embryos indicate the following: (a) afadin is not essential for processes earlier than egg cylinder formation; (b) afadin is not required for anterior–posterior body plan placement in egg cylinders; and (c) afadin is expressed specifically in the embryonic ectoderm, particularly the primitive streak region (neuroepithelium) and the neural fold/groove region, and plays an essential role in the junctional organization in the ectoderm during gastrulation.
To determine whether or not the defects in afadin−/−
embryos can be reproduced in a simpler model system, we took advantage of EB formation of ES cells where development of two-layered epithelial structures and subsequent mesoderm induction from the inner layer are shown to be reproduced in vitro (Doetschman et al. 1985
; Robertson 1987
; Rudnicki and McBurney 1987
). For this purpose, we first established an afadin−/−
ES cell line by introducing another targeting vector harboring puromycin-resistant gene ( A). This targeting vector was introduced into afadin+/−
ES cells (clone A46). ES cells were subjected to selection using puromycin. Southern blot analysis showed that three clones (B3, B8, and B103) resistant to puromycin underwent gene conversion, resulting in disruption of both alleles ( B). Western blot analysis using the anti–l-afadin (rat, aa 1814–1829) mAb and the mAb recognizing both l- and s-afadins (human, aa 1091–1233) revealed the loss of afadin in afadin−/−
ES cells and EBs ( C). Similar results were obtained with the polyclonal antibody recognizing both l- and s-afadins (rat, aa 577–592) (data not shown). Western blot analysis also revealed that l-afadin was a major expressed variant in wild-type ES cells and EBs, and that s-afadin was hardly detected ( C). Reverse transcription PCR analysis using the primer set corresponding to aa 188–316 of mouse afadin showed the loss of the afadin mRNA in afadin−/−
ES cells (data not shown). The three independent clones of afadin−/−
ES cells showed the same growth rate with undifferentiated morphology as wild-type ES cells did (data not shown). Since these clones showed the same phenotypes as far as examined, the data obtained from clone B3 were represented below.
Figure 7 Generation of afadin−/− ES cells. (A) Restriction maps of the afadin+/− allele, the second targeting vector, and the afadin−/− allele of the afadin gene. Filled boxes, exons. S, SacI; H, HindIII; (more ...)
When wild-type ES cells were subjected to suspension culture, cells aggregated to form EBs, some of which eventually develop to a two-layered cystic structure consisting of the outer endodermal layer and the inner high columnar ectodermal layer, although EBs with more complex structures with a large yolk sac–like cyst were often observed (, Aa and Ab). During earlier stages of EB formation, afadin−/− ES cells showed no significant difference (data not shown). Moreover, EBs with a large yolk sac–like cyst were often formed, suggesting that the endodermal components function normally ( Ba). On the other hand, in EBs with an amniotic cavity–like cyst, many necrotic cells were found in the cavity, while leaving the outer layer intact ( Bb). Generation of mesodermal cells at the space between the ectodermal and endodermal layers was observed, but the well-organized ectodermal layer did not develop. These results indicate that the ectoderm-specific defects of afadin−/− embryos may be reproduced in the in vitro EB model. Moreover, the presence of cellular components in the EB cavity may reflect the defects in the polarity of the ectodermal layer.
Figure 8 Disorganized ectoderm in cystic EBs derived from afadin−/− ES cells. Cystic EBs were subjected to histological analysis or immunofluorescence microscopy. For immunofluorescence microscopy, cystic EBs were doubly stained with rhodamine-phalloidin (more ...)
To investigate whether deficiency of afadin affects expression of other components of cell–cell junctions, we compared expression levels of E-cadherin, β-catenin, vinculin, ZO-1, and occludin, in wild-type and afadin−/− ES cells during EB formation. We could not detect any significant difference in their expression levels (data not shown).
Cytological Basis for the Defect of Afadin−/− EBs
EBs with an amniotic cavity–like cyst were selected and subjected to immunofluorescence microscopy using antibodies against various components of cell–cell junctions. Consistent with the results on embryos, cells in the ectodermal layer, but not in the outer endodermal layer of wild-type EBs expressed l-afadin ( Ac). However, unlike embryos with the high expression along the anterior–posterior axis, l-afadin was expressed ubiquitously in the ectodermal layer. l-Afadin was concentrated at the junctional complex regions where F-actin was concentrated, although diffuse distribution of F-actin along the entire cell surface was also observed (, Ac and Ad). In afadin−/− EBs, no afadin signal was observed ( Bc), indicating that the function of the gene is completely disrupted. Although no abnormality was found in the outer endodermal layer, formation of the organized junctional complex was severely inhibited in the ectodermal layer (, Bd–Bf). F-actin showed diffuse distribution along the entire cell surface without any concentration in the ectodermal layer ( Bd). Compared with the organized concentration of E-cadherin at the junctional complex regions in wild-type EBs ( Ae), E-cadherin was diffusely distributed over the ectodermal cell surface ( Be). Likewise, compared with the organized localization of ZO-1 in wild-type EBs ( Af), it was displayed as dotty signals in a random manner in the cell mass ( Bf).