The presented study demonstrates an important role of cortactin in early murine embryonic development during zygote maturation. This finding was unexpected in light of two recent reports about little or only a modest defect in the actin cytoskeleton reorganization in cortactin null MEFs [14
]. In particularly, a cortactin null MEF cell line generated by the Okabe’s group was successfully isolated from an embryo after intercrossing Cttn(−/+) mice [15
], implying that cortactin may not be required for early embryonic development. One possible explanation for the discrepancy is that different targeting systems were used to target the cortactin allele. The mice from Okabe’s group were derived from a CMV-Cre/loxp system, while ours were from an ES cell line carrying a gene trapping vector that was inserted in the intron between exon 7 and exon 8 (). Sequence analysis of a putative fusion transcript between cortactin exon 7 and the gene trapping vector predicts generation of a truncated protein that contains a cortactin N-terminal sequence from 1 to 157 amino acids. Thus, it is possible that the phenotype manifested by our cortactin knockout mice might be due to a dominant negative effect of this putative truncated protein. Although we have not yet been able to demonstrate the presence of the truncated protein product in Cttn(−/+) MEF using a cortactin polyclonal antibody against whole recombinant cortactin (data not shown), we prepared a GFP-Cttn(1–155) construct and analyzed whether this N-terminal truncated protein has any significant impact on NIH3T3 cells. Microscopic analysis of the transfectants revealed no significant alteration in either the actin cytoskeleton or apoptosis (Supplementary Fig. S4 and Fig. S5
), suggesting that the truncated protein has little dominant negative effect on cell growth. The insertion of the gene trapping vector may also create additional dominant negative protein products. Inspection of the murine cortactin genome sequence indicates fusion of three exones preceding exon 7 with the vector would generate in-frame transcripts. Indeed, a Western blot analysis of Cttn(−/+) MEF lysate using a β-galactosidase antibody revealed a protein band with an apparent molecular weight in the range of 155 to 160 KDa (data not shown). While we have not yet defined the precise form of this protein product, its gel motility agrees with a possible fusion between the β-neo gene and exon 3, which encodes the N-terminal 29 amino acids, a motif for binding to the Arp2/3 complex. We have previously shown that the function of this domain requires the whole F-actin binding motif to achieve an optimal binding to Arp2/3 complex [20
]. Since we did not observe any defect in the offspring production from intercrossing between Cttn(−/+) mice, which harbored a single copy of the mutated allele, this fusion protein product would likely have a minimal impact in vivo. However, we could not exclude the possibility that the fusion protein may affect zygote development in the context where no normal cortactin alleles are present. Because currently there is no cortactin homozygous mouse available, a final resolving of this issue would be beyond the limit of the system employed in this study.
We also noticed different genetic backgrounds involved in these studies, which may influence the final observed phenotype [31
]. The heterozygous mice generated by Okabe’s group were apparently a mixture of 129/sv-C57BL/6-Balb/c based on the information described in the paper. In contrast, the mice used in this study have a genetic background of 129/sv-C57BL/6. Whether a Balb/c background could compromise the defect caused by null cortactin in zygotic development will be tested in the future. Another explanation for the unexpected phenotype is that the gene trapping vector in our mice may disrupt an allele for another essential gene of which the identity is unknown. If this is the case, one would expect that this allele has to be in a position very close to that of cortactin so that it would be co-segregated with the cortactin allele all the time. So far, we have not been able to detect multiple copies of the vector in the genome of the mice based on a quantitative PCR analysis (data not shown). Future study using fluorescence in situ hybridization may be required to vigorously rule out this possibility.
We conclude that cortactin plays a more vital role in oocytes than somatic cells. There is another case for a paramount role of actin cytoskeleton proteins in oocytes. Complete knockout of formin-2, which is an actin nucleator, did not affect mice development but influenced the maturation of MI-arrested oocytes [32
]. Formin-2-deficient oocytes failed to extrude the first polar body and were impaired in the migration of meiotic spindle toward the polarized cortex at the first meiosis stage. However, formin-2 deficient oocytes retained the ability to form the actin cap [33
], indicating that the formation of the actin cap is a formin-2 independent event. We provided evidence here that cortactin-mediated actin polymerization is responsible for the actin cap formation. First, we observed that cortactin overlaps well with the actin cap (). Furthermore, a chemical inhibiting actin polymerization abolished the polarization of cortactin. Also, microinjection of two types of cortactin antagonists inhibited the formation of the actin cap ( and ). The finding for the role of cortactin in the actin cap implies that different types of actin filaments execute distinct functions in the asymmetric division. It is well established that formin proteins direct formation of linear actin filaments in somatic cells [34
]. In oocytes, formin-2 has been thought to be responsible for the formation of actin clusters that are transiently organized into stress fibers [35
]. In contrast, cortactin participates in the Arp2/3 mediated-actin polymerization, which is characterized by branched actin filaments. Thus, our data strongly suggests that the actin cap is made of branched actin filaments. Enrichment of branched actin filaments in the actin cap may provide a structural explanation for a previously well-described observation that the membrane domain for the actin cap is normally devoid of microvilli [2
], a type of membrane protrusion that is primarily filled with straight actin bundles. The exact function of the actin cap in oocytes remains not fully defined. In addition to the asymmetric division, it has been also suggested that the actin cap is involved in the direction of migrating chromosomes during the first meiosis [1
]. In the present study we have only looked at MII-oocytes where the chromosome has already been located to the proximity of the cortex where the first polar body is extruded. Therefore, the role of cortactin in the chromosome migration is not clear, and we will address this issue with MI-oocytes in the future. However, in the analysis of MII-oocytes injected with a cortactin mutant deficient in Arp2/3 binding, we have found a dislocation of the maternal chromosome in relation to the polar body (). This suggests that cortactin may be required for the stabilization of the chromosome in the cortex of MII-oocytes.
Our microinjection experiment using cortactin antagonists indicated a role for cortactin in the asymmetric division. While we could not rule out the possibility that these antagonists may have some unexpected side-effects, cortactin could play other roles in egg activation. In fact, we have observed a gradual decrease in the detection of homozygous zygotes after fertilization. The precise reason for the gradual loss of homozygous oocytes is unknown. Since the majority of homozygous eggs displayed certain morphological abnormality such as defragmentation and many eggs with the abnormal morphology failed to give results in the PCR based genotyping, it was quite possible that many cortactin homozygous embryos had become fragile or undergone apoptosis during either in vivo or in vitro process. How the loss of cortactin could make oocytes more fragile is unknown. Because of the difficulty to obtain homozygous embryos and the presence of parent proteins, the effort to characterize in more detail the function of cortactin in the egg activation will be made with other animal models such as conditional knockout mice.