The nuclear-cytoplasmic distribution of the Xenopus nuclear factor, xnf7, coincides with its state of phosphorylation during early development

An important question concerns the mechanism by which the embryo regulates the function of maternal gene products that may not be required until specific developmental stages such as the MBT (Newport and Kirschner, 1982). In Xenopus, there are a number of maternal gene products that exhibit interesting patterns of behavior during early development that may indicate a role of compartmentalization and post-translational modification in regulating their function. Dreyer (1987, 1989) described a number of antigens that originate in the oocyte nucleus (GV), become cytoplasmic during oocyte maturation, and re-enter the embryonic nuclei at specific times during development. Some re-enter immediately following fertilization while others reenter either at the blastula or neurula stages of development.

Recently, we have been studying in Xenopus the gene xnf7 whose protein product originates in the oocyte GV, becomes cytoplasmic at maturation and is retained in the cytoplasm until it re-enters the embryonic nuclei by the mid-blastula stage (Dreyer et al. 1983; Miller et al. 1989; Kloc er al. 1989; Reddy er al. 1991). It ultimately becomes enriched in nuclei of the central nervous system at larval stages of development. Xnf7 also binds to double-stranded DNA and is detected associated with lampbrush chromosomes (Miller er al. 1989; D. Wedlich, unpublished observations). We have also shown that the conceptual xnf7 protein possesses several zinc-finger-like regions, two acidic regions that are similar to transcriptional activating domains, and a p34^cdc2 protein kinase phosphorylation site (Reddy et al. 1991). In addition, it is an in vitro substrate for p34^cdc2 protein kinase. Recently, we have shown that one of the acidic domains can transactivate a reporter gene in a transfection assay (B. Reddy and L. Etkin, unpublished observations). These results suggest that the xnf7 protein may function as a transcription factor during development.

In the present study, we were interested in determining several of the characteristics of the xnf7 protein that may help us to define further its behavior and understand its function during development. We report here that (1) the xnf7 protein is phosphorylated at maturation resulting in the formation of slowly migrating isoforms, (2) the phosphorylated slow isoform is in the cytoplasm of maturing oocytes, (3) the phosphorylated slow isoform predominates during early cleavage when the protein is cytoplasmic while the fast migrating isoform becomes predominant again by the midblastula stage when the protein is accumulating in nuclei, and (4) the xnf7 protein possesses a putative nuclear localization signal (NLS) similar to that of nucleoplasmin and can efficiently enter the oocyte GV within a short time following synthesis. These results suggest that both phosphorylation and cytoplasmic sequestration are involved in regulating the function of xnf7 during early development. They also suggest that the xnf7 protein may be retained in the cytoplasm during early development by interfering with the nuclear localization mechanism.

Xenopus laevis were obtained from Nasco (Wisconsin). Frogs and tadpoles were maintained according to Gurdon (1967). Ovulation was induced by injection of 800i.u. human chorionic gonadotropin (hCG) (Sigma) into the dorsal lymph sacs of females. Fertilization was performed either by spawnings where males were injected with 300i.u. hCG or by in vitro techniques (Etkin et al. 1984; Tompkins, 1978).

GVs were manually isolated in 1×Modified Barth’s Saline (MBS). Oocytes were either manually defolliculated in MBS or defolliculated by incubation in 50 mg ml^-1 collagenase (type IV; Sigma) in calcium- and magnesium-free OR-2 (Wallace et al. 1973) at 25– 30°C for 45– 90 min. Maturation of defolliculated oocytes was induced by culturing in 1×MBS with 10μgml^-1 progesterone (Sigma). Maturation (GVBD) was determined by the presence of a white spot in the animal pole or by dissection of oocytes. To study phosphorylation, oocytes were incubated in 0.5mCiml^-1 inorganic ³²P-phosphate (carrier-free; Amersham) for 4h in 1× MBS. The free label was removed by extensive washing with 1×MBS and oocytes were matured with progesterone. Ovulated eggs were dejellied with 2.5 % cysteine in 50 mM Tris– HCl (pH 7.8) and extensively washed with 1× MBS. Protein extracts were prepared by homogenization of GV, oocytes, and eggs in a buffer consisting of 10 mM Tris– HCl (pH 8.0), 10 mM DDT, 0.1% Triton X-100 and 5mM EDTA. Protease inhibitors included 0.1– 1 mM PMSF, 0.5μgml^-1 leupeptin and 0.7μgml^-1 pepstatin. Phosphatase activity was inhibited by the addition of 50mM β-glycerophosphate (Sigma). Protein concentration determinations of extracts were performed using the Bio-Rad Protein determination kit with BSA as a standard.

Immunoprecipitation was performed according to Anderson and Blobel (1983) using 1:50 diluted antisera and protein A– sepharose beads (Pharmacia) as a solid phase immunoad-sorbant. Samples were separated electrophoretically under denaturing conditions (Dreyfuss et al. 1984; Laemmli, 1970). Western blots were performed either as in Miller et al. (1989) or using a semidry Sartoblot apparatus (Sartorius, Hayward, CA) as recommended by the manufacturer. Two antibodies were used, one was the monoclonal antibody, 37-1A9, prepared from Xenopus GV proteins (Dreyer et al. 1981) and a second antibody, the polyclonal antibody (L24), produced from the bacterially expressed xnf7 protein, produced identical results (Reddy et al. 1991). The preparation of monoclonal and polyclonal antibodies and the developmental fates of their antigens is described elsewhere (Dreyer et al. 1981, 1982, 1983, 1985; Reddy et al. 1991).

Samples were combined with an equal volume of 0.2 M Tris–HCl (pH9.5), 0.2M NaCl and 10mM MgCl₂ and incubated at 37°C for 0– 18h with 0.01– 2mgml^-1 bacterial alkaline phosphatase (BAP) (Sigma). Effective concentrations of BAP were determined experimentally.

Embryos, tadpoles and portions of adult tissues were fixed in 100% methanol, overnight, at 4°C, dehydrated with 100% ethanol, cleared in xylene, embedded in paraplast and cut into 5 μm sections. Sections were treated with xylene to remove the paraplast. For immunofluorescent staining, the nonspecific protein-binding sites were blocked by incubation in blocking solution (3% BSA, 10% goat serum in PBS) for 30min– 1h (Wedlich and Dreyer, 1988). Subsequently, sections were incubated for 1 h with the 37-1A9 monoclonal antibody (1:500 in blocking solution) and washed two times (10min each) in PBS. Fluorescein–isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (BMB) (1:20 to 1:2000 dilution in PBS) was used as a secondary antibody. After 1 h incubation sections were washed in PBS (two times, 15 min each) and mounted in 50% glycerol in PBS.

Stage 6 oocytes (Dumont, 1972) were labeled with [³⁵S]meth-ionine (specific activity-1000 Ci mmol^-1; Amersham) in MBS during which samples were taken at various time points. At each time point, 25 oocytes were manually dissected into cytoplasms and GVs, the protein was extracted, and xnf7 was immunoprecipitated using the L24 polyclonal antibody. The immunoprecipitated protein was analyzed by PAGE and fluorography.

We previously reported the presence of a fast-migrating isoform of xnf7 in oocytes and a slow-migrating form present in eggs (Miller et al. 1989). The presence of a fast isoform in oocytes and a slow isoform in eggs suggested that the slow isoform was generated during oocyte maturation and may be the result of phosphorylation. This was tested by maturing oocytes in vitro with progesterone and analyzing samples taken during maturation by western blot.

Fig. 1 shows that there is a dramatic shift in the isoforms from the faster oocyte form to the more slowly migrating egg forms in oocytes that were matured in vitro. No shift to the egg isoforms was observed in oocytes cultured in the absence of progesterone. The peak shift to the egg isoforms occurred at 0.6–0.8 GVBD₅₀ in which 0 GVBD₅₀ is the time of progesterone addition and 1.0 GVBD₅₀ is the time when 50% of the oocytes have undergone GVBD. With the appearance of the slower egg isoform, there was a concomitant decrease in the amount of the fast oocyte isoform. This result shows that during the process of oocyte maturation new xnf7 isoforms are generated. It is likely that the new isoforms were generated from pre-existing protein since the rate of synthesis of xnf7 is too low to produce enough new protein during maturation to be detected on western blots.

The change in mobility of xnf7 that we observed at maturation is typical of proteins that have undergone phosphorylation. Therefore, we performed two different experiments to determine whether xnf7 was phosphorylated during maturation and that phosphorylation accounted for the generation of the slow-migrating xnf7 isoform.

In the first set of experiments, defoiliculated oocytes were incubated 4h in 1×MBS with 0.5mCiml^-1 carrier-free ³²P-inorganic phosphate. After washing away the free label, oocytes were cultured 4–8 h in 1× MBS in the presence or absence of progesterone and scored for maturation. Immunoprecipitates of the extracts prepared from matured (egg) and non-matured oocytes showed that, while the oocyte samples incorporated low levels of label into xnf7, much more label was incorporated into xnf7 in the egg samples (Fig. 2A). Western blots showed that the immunoprecipitated radioactive xnf7 from the eggs and oocytes comigrated with the egg and oocyte isoforms, respectively (data not shown). The radioactive faster oocyte isoform is not detectable in Fig. 2A due to the overshadowing of the band by the large amount of label in the slower egg isoform.

In the second set of experiments, extracts prepared from oocytes and eggs were treated with BAP to substantiate further the role of phosphorylation in the production of the xnf7 isoforms. Fig. 2B shows an immunoblot of oocyte and egg extracts treated with BAP. The BAP treatment did not affect the mobility of the fast oocyte isoform; however, it did produce a shift in migration of the slow egg isoforms to the fast oocyte isoforms (lanes 2 and 4). This confirms that phosphorylation of the oocyte isoforms results in their reduced mobilities on gels and clearly demonstrates that the isoforms of xnf7 produced during oocyte maturation are due to phosphorylation.

We were interested in determining whether the new phosphorylated isoforms of xnf7 produced during maturation were located in the nucleus or the cytoplasm. Therefore, we treated defoiliculated oocytes with progesterone and at subsequent times prepared extracts from the GVs, enucleated cytoplasms and intact oocytes.

Fig. 3 shows that the majority of xnf7 was localized in the GV with a small amount in the enucleated oocyte cytoplasm from the time of progesterone addition until 150min later. This is consistent with observations using immunocytochemistry (Dreyer et al. 1983). Interestingly, the small amount of xnf7 detected in the cytoplasm prior to 150min was the fast oocyte isoform. The onset of the major shift to the slow egg isoforms began at 150 min after progesterone addition which was well before GVBD_50. Furthermore, after 150min, it was the slow egg-specific isoform that was present in the cytoplasm while the antigen present in the intact GV was the fast oocyte isoform. By 210min, roughly 50% of all oocytes had undergone GVBD and it was impractical and difficult to isolate intact GVs. These data show that during oocyte maturation a small amount of xnf7 is found in the cytoplasm before GVBD and that prior to 150 min it all remains in the hypo-phosphorylated fast oocyte form. After 150min (but prior to GVBD), however, the phosphorylated cytoplasmic slow-migrating egg form of xnf7 appears.

Previous studies have shown that xnf7 is retained in the cytoplasm until it reenters the nuclei by the mid-blastula to gastrula stages of development (Dreyer et al. 1983). We were interested in determining the distribution of the different isoforms during development. Fig. 4 shows a western blot of protein extracts prepared from embryos of different developmental stages. During cleavage until stage 8 the predominant form was the slow-migrating egg-specific isoform, though a small amount of the fast-migrating form appears at stage 3. By stage 8–9 the fast-migrating oocyte isoform becomes predominant. Small amounts of the slow egg isoforms were still detected as late as early tailbud stages although their abundance decreased 2- to 3-fold. We also examined the time at which xnf7 reenters the nuclei by performing immunofluorescence on histological sections of early blastula (stage 7) and early gastrula (stage 10) embryos. Fig. 5 shows that xnf7 was not detectable in stage 7 nuclei; however, at stage 10 almost all nuclei showed staining. More importantly, the accumulation of xnf7 protein in the nuclei between stage 7 and 10 correlates well with the predominance of the faster migrating oocyte-like isoform (Fig. 4). Since we have shown that these isoforms differ in their degree of phosphorylation, this result suggests that the state of phosphorylation of xnf7 changes during development coincident with its transport from the cytoplasm to the nucleus.

One possible explanation for why the protein does not accumulate in nuclei until the blastula stage is that the xnf7 protein possesses a NLS that functions poorly, resulting in a slow accumulation of the protein in the nuclei. The conceptual sequence of the xnf7 protein shows the presence of a putative NLS signal KRKIEE-PEPEPKKAK located between amino acids 106 and 120 (Reddy et al. 1991). This is very similar to the bipartite NLS, KRPAATKKAGQAKKKK, found in nucleoplasmin (Robbins et al. 1991). As a preliminary assessment of the ability of xnf7 to enter nuclei, we performed the following experiment.

Newly synthesized xnf7 protein was labeled by incubation of stage 6 oocytes in [³⁵S]methionine during which samples of GVs and cytoplasms were taken. Protein was extracted and xnf7 was immunoprecipitated with the L24 polyclonal antibody (Reddy et al. 1991) and analyzed by PAGE and fluorography. We calculated the relative concentration of the protein in the GV and cytoplasm based on densitometry measurements of the bands on the fluorograph and the differential volume of the two compartments. Fig. 6 shows that the xnf7 protein was detected in the GV 5h following initiation of labeling (Lane 1). Based on the 8fold difference in volume of the GV and cytoplasm (Burglin and DeRobertis, 1987), we calculated that the xnf7 protein was 4-fold more concentrated in the GV than cytoplasm at this time point. At 15 h (Lanes 3 and 4) the protein was 8-fold more concentrated and at 30 h (lanes 5 and 6) 11 times more concentrated in the GV,

We calculated the lag time between the detection of xnf7 in the cytoplasm and its accumulation in the nuclei from this and two other experiments. The results indicated that the translocation time from the cytoplasm to the nucleus forxnf7 was2h. This translocation time is consistent with the immunofluorescent data (Fig. 5) showing that the xnf7 protein accumulated in nuclei during the several hour period between stages 7 and 10. These data show that the xnf7 protein possesses an efficient NLS and suggest that the maternal protein present at fertilization does not enter nuclei prior to the blastula stage due to a process that interferes with the function of the NLS.

We report that a nuclear protein (xnf7) that is enriched in the adult central nervous system of Xenopus is phosphorylated during oocyte maturation following its release into the cytoplasm. The phosphorylation results in the formation of several isoforms of the protein. Xnf7 is retained in the cytoplasm until the mid-blastula stage when it accumulates in the nuclei at detectable levels. The reaccumulation in nuclei is accompanied by a concurrent dephosphorylation of the xnf7 protein. The xnf7 protein is efficiently accumulated in oocyte GVs suggesting that its retention in the cytoplasm during early development may be due to a mechanism that interferes with the function of the NLS.

The conceptual xnf7 protein contains several putative zinc fingers, acidic domains similar in structure to activation domains found in numerous transcription factors (one of which is capable of activating a reporter gene), and three potential p34^cdc2 protein kinase phosphorylation sites (Reddy et al. 1991). Xnf7 binds to double-stranded DNA (Miller et al. 1989) but not to single-stranded DNA (C. Dreyer, unpublished observations) and is detected on lampbrush chromosomes suggesting an in vivo association with DNA (D. Wedlich and C. Dreyer, unpublished observations). In addition, the bacterially produced protein from the xnf7 cDNA as well as the endogenous xnf7 from the oocyte reacts with a polyclonal antibody made from the Krüppel finger link region ( both xnf7 and Krüppel have a common epitope recognized by the antibody, B. Reddy, C. Dreyer and L. Etkin, unpublished observations). These data, taken together, support the conclusion that xnf7 is a DNA-binding protein and may function as a transcription factor.

Our evidence for phosphorylation of xnf7 during oocyte maturation includes the shift in gel mobility of xnf7, incorporation of inorganic ³²P into immuno-precipitable xnf7 and the removal of phosphate groups with bacterial alkaline phosphatase (BAP). Additionally, we have shown that bacterially produced xnf7 is phosphorylated by MPF (Reddy et al. 1991). The removal of phosphate groups with BAP converts the egg isoforms into species that co-migrate with the oocyte isoforms.

We have shown that xnf7 is an in vitro substrate for a cell-cycle-specific kinase, MPF and have mapped the site of phosphorylation to a putative p34^cdc protein kinase consensus site (Reddy et al. 1991; Gautier et al. 1988; Moreno and Nurse, 1990). Our data demonstrating the phosphorylation of xnf7 by MPF in vitro and the observation that xnf7 is phosphorylated during oocyte maturation suggest that it may also be an in vivo substrate (Reddy et al. 1991).

There is also a close correlation between the state of phosphorylation of xnf7 and its accumulation in nuclei during cleavage. The embryonic relocalization of xnf7 during development correlates with the increased accumulation of the faster moving (lower state of phosphorylation) oocyte isoform. The observation that both faster and slower migrating isoforms co-exist in embryos reflects that the shift from the cytoplasm to the nucleus is not an all- or-none phenomenon, but takes place over a period of embryonic development culminating in the highest levels of accumulation at the midblastula stage. This is supported by immunohistochemical data showing that oocyte isoforms of xnf7 are nuclear, while the egg forms are largely cytoplasmic during early development. It was found that the nuclear transport kinetics of the SV40 T antigen were effected by sequences outside of the nuclear transport signal that are sites of potential phosphorylation (Rihs and Peters, 1989). Interestingly, in xnf7, the major site for in vitro phosphorylation actually overlaps the putative NLS suggesting that phosphorylation of this site may affect the functioning of the NLS (Reddy et al. 1991). However, our understanding of the relationship between phosphorylation and nuclear/cytoplasmic partitioning for xnf7 will have to await the results of further studies.

There are several examples of temporally and spatially specific movements of proteins from the cytoplasm to the nucleus during development and cell differentiation at which time they are functional. An example is the Drosophila dorsal gene product which encodes a transcription factor required for the development of ventral structures (Hunt, 1990; Roth et al. 1989; Rushlow et al. 1989; Steward, 1989). Dorsal appears to function by activating the transcription of at least two genes, twist and snail, that are expressed in the ventral region of the embryo. Interestingly, during normal development the dorsal gene product is present in all regions of the embryo, but in the ventral region it is localized in the nuclei, and in the dorsal region in the cytoplasm. The difference in nucleocytoplasmic compartmentalization of the dorsal gene product may be due to its interaction with the cactus gene product resulting in anchoring of the dorsal protein in the cytoplasm. This interaction may be augmented by differential phosphorylation of the dorsal protein in different regions of the embryo, which may affect its binding to the cactus protein (Hunt, 1990). Another protein whose function may be regulated by its nuclear-cytoplasmic distribution is the transcription factor NF-kB, which functions in the activation of the kappa chain gene in B lymphocytes (Lenardo and Baltimore, 1989). In unstimulated cells, this protein is complexed with another protein I-Kb resulting in sequestration in the cytoplasm and inability to translocate into the nucleus. The interaction appears to be dependent on the phosphorylation of the I-kB protein. In Xenopus, the c-myc protein is retained in the cytoplasm in the oocyte and becomes nuclear following fertilization suggesting a possible association with a cytoplasmic anchor protein in oocytes (Gusse et al. 1989).

The xnf7 protein becomes cytoplasmic at oocyte maturation, is retained in the cytoplasm as a phosphorylated form, and accumulates in nuclei by the midblastula to gastrula stages of development. We do not believe that the delay in accumulation of the xnf7 protein during development is due to an inefficient nuclear translocation signal since our data show that newly synthesized endogenous xnf7 protein accumulates in the GV efficiently with kinetics similar to proteins such as nucleoplasmin that are detected in embryonic nuclei immediately following fertilization. We also have preliminary data suggesting that the xnf7 protein associates with large protein complexes during cleavage stages when it is cytoplasmic (X. Li and L. Etkin, unpublished observations). These data suggest that the xnf7 protein may be actively sequestered in the cytoplasm during early cleavage stages, perhaps by interaction with other proteins until it is needed at or soon after the mid-blastula stage. We propose that the compartmentalization in the cytoplasm and post-translational modification of proteins such as xnf7 are important mechanisms by which Xenopus regulates the function of nuclear proteins during cleavage stages prior to the MBT.

This work was supported by grants DCB-8608690 and DCB-9007410 from the National Science Foundation to L.D.E. and a postdoctoral training fellowship (CA09299) to M.M. We would like to thank Dr Jian Kuang for critical comments on the manuscript.