Neuronal protein GAP-43 in early mouse embryos

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Background

Growth-associated protein 43 (GAP-43) is a specific neuronal protein found in vertebrate species. In neurons, this protein is predominantly associated with the plasma membrane of axon terminals [1–3]. GAP-43 plays an important role in the embryonic development of the nervous system, participating in axonal growth cone guidance. In adult organisms, GAP-43 supports synaptic plasticity and regenerative processes in the nervous system [4–9]. At the molecular level, GAP-43 clusters acidic phosphoinositides in the plasma membrane via its polycationic effector domain under the control of calmodulin and protein kinase C, thereby participating in the regulation of the actin cytoskeleton dynamics [10, 11]. GAP-43 function is presumably mediated by the formation of large oligomer complexes in the presence ofacidic phospholipids [12, 13]. GAP-43 belongs to a group of natively disordered proteins, which are characterized by a variety of protein–protein interactions and multiple functions [13]. For a long time, GAP-43 was considered neuron-specific [1, 7]. However, there is currently evidence of GAP-43 presence in glial cells, chromaffin cells [1], podocytes [14], muscle [15], and cancer cells [16]. We have recently shown that GAP-43 is also present in mouse oocytes and zygotes. In metaphase II oocytes, GAP-43 is localized in tubulin-based structures — the meiotic spindle and microtubule-organizing centers — and phosphorylated by protein kinase C [17]. Notably, the expression, modifications and functions of GAP-43 vary significantly across different cell types, whereas its interactions with the main intracellular partners — protein kinase C, calmodulin, actin, and acid phospholipids — are preserved [17]. GAP-43 has also been detected in proliferating pluripotent cells (mouse embryonic carcinoma cells) [18] and multipotent cells (neuronal precursors), where it is localized in the cytoplasm, on centrosomes, and in the chromatin region [19].

The aim of the study was to investigate the localization of GAP-43 in toti- and pluripotent cells of early mouse embryos.

Materials and Methods

C57BL/CBA F1 hybrid mice were used in the study. To obtain oocytes and zygotes, female mice were hormonally stimulated by administering 10 IU of pregnant mare serum gonadotropin (Mosagrogen, Russia) followed by 10 IU of human chorionic gonadotropin (Intervet International B.V., Netherlands) after 42–46 hours. The animals were euthanized by cervical dislocation 13–20 hours after the administration of human chorionic gonadotropin. The oocyte/zygote–cumulus cell complexes were then collected from the mouse oviduct. The mouse embryos were cultured up to the blastocyst stage in a CO2 incubator (5% CO2, 37 °C) in G-1 Plus medium (Vitrolife, Sweden).

Immunocytochemical staining of oocytes and early embryos was performed using a standard protocol [17]. The anti-GAP-43 primary antibodies AB5312 and AB5401 (Millipore, USA) were used at a 200-fold dilution. The secondary antibody, A-11034, conjugated with the Alexa Fluor 488 fluorescent dye (Invitrogen, USA) was used at a 500-fold dilution. Chromosomes were stained with DAPI. The immunostained oocytes and embryos were mounted on glass slides using the Vectashield mounting medium (Vector Laboratories, USA) and analyzed using an LSM-510 Meta confocal microscope (Carl Zeiss, Germany) at the Resource Center for the Development of Molecular and Cellular Technologies (St. Petersburg State University, Russia).

Results and Discussion

Previously, to study GAP-43 in mouse oocytes and zygotes, we used specific polyclonal antibody against rat GAP-43, AB5220, and AB5401 against the phosphorylated (at Ser41) form of GAP-43 (Millipore, USA) [17]. In this study, the distribution of GAP-43 in metaphase II oocytes and early mouse embryos, from the single-cell stage (zygote) to the blastocyst stage, was studied using immunocytochemical staining with polyclonal antibody against cat GAP-43, AB5312 (Millipore, USA).

In oocytes, a uniform distribution of GAP-43 is observed throughout the cytoplasm, with the highest intensity of staining in the meiotic spindle region (see Fig. 1, a). In zygotes, GAP-43 is present in the pronuclei (except for the nucleoli) and cytoplasm. Notably, the pronuclei generally exhibit a higher intensity of staining compared to the cytoplasm (see Fig. 1, b). In 2-cell and 4-cell stage embryos, a similar pattern is observed: GAP-43 is evenly distributed throughout the blastomere cytoplasm and is present in the nuclei, but not in the nucleoli (see Fig. 1, c, d).

 

Fig. 1. Localization of GAP-43 in oocytes and early mouse embryos: a, metaphase II oocyte (arrow indicates the position of the meiotic spindle); b, interphase stage zygote (pb, the second polar body); c, 2-cell stage embryo; d, 4-cell stage embryo. DIC, differential interference contrast; DAPI, DNA staining (blue); AB5312, GAP-43 staining with AB5312 antibodies (green); Merge, DAPI and AB5312 combined

Рис. 1. Локализация белка GAP-43 в ооците и ранних эмбрионах мыши: a — ооцит на стадии метафазы II (стрелкой указано положение веретена с хромосомами); b — зигота на стадии интерфазы (pb — второе полярное тельце); c — эмбрион на стадии двух бластомеров; d — эмбрион на стадии четырех бластомеров. DIC — дифференциально-интерференционный контраст, DAPI — окрашивание ДНК, AB5312 — окрашивание GAP-43 антителами AB5312, Merge — совмещение каналов DAPI и AB5312

 

At the morula stage, GAP-43 is also localized in the cytoplasm and nuclei (see Fig. 2, a, b). In some blastomeres, GAP-43 staining intensity is higher than in other blastomeres of the same embryo. Fig. 2a shows a morula at the beginning of compaction, with two of eight blastomeres having more intense GAP-43 staining. This probably indicates that GAP-43 expression in early embryos is dynamical and depends on the phase of the cell cycle. Consequently, due to the asynchronous division of blastomeres, the amount of GAP-43 in different blastomeres may vary.

 

Fig. 2. Localization of GAP-43 in early mouse embryos: a, 8-cell stage morula; b, late morula; c, early blastocyst; d, hatching blastocyst (ICM, inner cell mass, tb, trophoblast cells). DIC, differential interference contrast; DAPI, DNA staining (blue); AB5312, GAP-43 staining with AB5312 antibodies (green); Merge, DAPI and AB5312 combined

Рис. 2. Локализация белка GAP-43 в ранних эмбрионах мыши: a — морула на стадии восьми бластомеров; b — поздняя морула; c — ранняя бластоциста; d — бластоциста на стадии вылупления (ICM — клетки внутренней клеточной массы, tb — клетки трофобласта). DIC — дифференциально-интерференционный контраст, DAPI — окрашивание ДНК, AB5312 — окрашивание GAP-43 антителами AB5312, Merge — совмещение каналов DAPI и AB5312

 

At the next stage, the blastocyst, the embryo cells differentiate into the trophoblast (the outer layer of cells of the blastocyst) and the inner cell mass, which later forms a pluripotent epiblast. Fig. 2, c shows an early blastocyst with a forming cavity. It is notable that the intensity of GAP-43 staining in the nuclei is higher than that in the cytoplasm. In the blastocyst at a later stage (during hatching from the zona pellucida), a more intense staining of the inner cell mass is clearly visible compared to the trophoblast cells (see Fig. 2, d).

In this study, we described for the first time the expression of GAP-43 in early mouse embryo cells, from the zygote to the blastocyst stage. In early embryos, GAP-43 exhibits cytoplasmic and nuclear localization, which differs significantly from the predominantly membrane localization of GAP-43 in neurons. Intense staining of the cytoplasm with anti-GAP-43 antibodies is observed in embryos up to the 4–8-cell stage. GAP-43 is also localized in the zygote pronuclei and early embryo nuclei. Thus, the nuclear localization of GAP-43 was demonstrated for the first time. The presence of GAP-43 in the nucleus was also confirmed by antibody AB5401 against the Ser41-phosphorylated form of GAP-43 (Millipore, USA) and anti–rat brain GAP-43 monoclonal antibody G9264 (Sigma-Aldrich, USA). Both antibodies also stain cell nuclei in early mouse embryos (see Fig. 3; for G9264, data are not provided). Staining of the nuclei and cytoplasm with AB5401 indicates that in early embryos GAP-43 is phosphorylated, presumably by protein kinase C as in neurons.

 

Fig. 3. Localization of Ser41-phosphorylated GAP-43 (pSer41-GAP-43) in morula. DIC, differential interference contrast; DAPI, DNA staining (blue); AB5401, pSer41-GAP-43 staining with AB5401 antibodies (green); Merge, DAPI and AB5401 combined

Рис. 3. Локализация белка GAP-43, фосфорилированного по остатку Ser41, в моруле. DIC — дифференциально-интерференционный контраст, DAPI — окрашивание ДНК, AB5401 — окрашивание pSer41-GAP-43 антителами AB5401, Merge — совмещение каналов DAPI и AB5401

 

It is well known that the membrane localization of GAP-43 in neurons is associated with palmitoylation of its two residues, Cys3 and Cys4 [20, 21]. Therefore, it can be concluded that GAP-43 is not palmitoylated in early mouse embryos. As we suggested earlier, the absence of palmitoylation may be explained by the expression of the short isoform of GAP-43 (GAP-43-2), lacking residues 1–4 and truncated at the N-terminus due to the alternative translation initiation from the Met5 codon [17]. We analyzed the amino acid sequence of GAP-43 using computer algorithms designed to predict the subcellular localization of the protein. The PSORT II [22], SCLpred [23], ESLPred2 [24], and Cello v.2.5 [25] programs predict nuclear localization of GAP-43. The MultiLoc2 [26] and BUSCA [27] programs predict nuclear/cytoplasmic localization of GAP-43. The cNLS Mapper [28] and NLStradamus [29] programs predict that the polycationic effector domain of GAP-43 contains a monopartite nuclear localization signal (residues 43–57). It is interesting to note that the two GAP-43-related proteins, MARCKS and BASP1, have also been shown to contain a nuclear localization signal in their effector domains [30, 31]. Thus, the prediction algorithms confirm the nuclear and cytoplasmic localization of unpalmitoylated GAP-43 revealed in the present study.

Comparing the data obtained using two polyclonal antibodies against GAP-43, AB5312 (this study) and AB5220 (previous study [17]), two differences can be noted. (1) AB5312 produce more intense staining of the cytoplasm than AB5220. Consequently, staining of tubulin-based structures (the meiotic spindle and microtubule-organizing centers) with AB5312 is not as pronounced, although staining of the spindle region is still more intense than staining of the cytoplasm (see Fig. 1, a). (2) Unlike AB5220, AB5312 stains cell nuclei and zygote pronuclei. Thus, AB5220 and AB5312 have different specificities to the nuclear and cytoplasmic pools of GAP-43. Both AB5220 and AB5312 are rabbit polyclonal antibodies, but their immunogens are different: they were raised against rat and cat GAP-43, respectively. Approximately 20% of amino acid residues in the rat and cat GAP-43 sequences are different, which may result in different epitopes for these antibodies.

Based on the results of this and previous [17] studies, three different intracellular pools of GAP-43 can be distinguished in mouse oocytes and early embryos: (a) cytoplasmic, (b) nuclear, and (c) associated with tubulin-based structures (the meiotic spindle and microtubule-organizing centers). Moreover, our preliminary data show that these pools are selectively stained with different anti-GAP-43 monoclonal antibodies (the manuscript is being prepared for publication). This is probably due to various modifications of GAP-43 in these pools, which will be investigated in future work.

Early (preimplantation) embryos attract special interest due to the unique properties of their cells — toti- and pluripotency. In placental mammals, totipotency (the potential to develop into a complete organism from a single cell) includes the ability to generate both the embryo and the extraembryonic tissues. For mouse embryos, it has been shown that only the zygote and a single blastomer of the 2-cell and 4-cell embryos are totipotent [32]. Blastomeres of mouse embryos at later stages of development no longer possess this property. At the late blastocyst stage (before implantation into the uterus), a subset of cells within the inner cell mass forms the pluripotent epiblast. Epiblast cells are capable of differentiating into cells of all three embryonic germ layers; however, they are not capable of forming extraembryonic tissues. The epiblast is a source for obtaining pluripotent embryonic stem cells in vitro. In this study, GAP-43 was detected in mouse totipotent and pluripotent embryonic cells. In this regard, it should be noted that there are several reports on the expression of GAP-43 in pluripotent cells of mouse embryonic carcinoma. In these cells, GAP43 gene transcription was found to be initiated from a promoter different from the one responsible for GAP43 transcription in neurons [18, 33]. GAP-43 has also been detected in human embryonic stem cells [34] and cancer cells [16]. This suggests that GAP-43 plays a special role in undifferentiated cells.

Conclusion

In this study, we demonstrated for the first time the presence of GAP-43 in early mouse embryo cells, from the zygote to the blastocyst stage. In early embryo cells, GAP-43 is localized in the cytoplasm and nucleus, unlike in neurons, where it is predominantly associated with the plasma membrane. Since the putative role of GAP-43 in neurons is related to the regulation of the dynamics of the plasma membrane and actin cytoskeleton, our findings suggest specific functions of GAP-43 in toti- and pluripotent cells of early embryos. Our findings are also important for studying the role of GAP-43 in other undifferentiated (stem and cancer) cells.

Additional information

Funding source. This study was not supported by any external sources of funding.

Ethics approval. The protocol of the study was approved by the local Ethics Committee of the Institute of Experimental Medicine № 2/22 dated 2022 April 6.

Competing interests. The authors declare that they have no competing interests.

Author contribution. All authors made a substantial contribution to the conception of the study, acquisition, analysis, interpretation of data for the work, drafting and revising the article, final approval of the version to be published and agree to be accountable for all aspects of the study.

Personal contribution of each author: F.M. Zakharova, V.V. Zakharov — concept and design of the study, analytical processing of the results, writing the text of the article; F.M. Zakharova, N.A. Yagovkina — working with animals, immunocytochemical experiments, data analysis; F.M. Zakharova — working on a confocal microscope; V.V. Zakharov — working with databases and programs.

About the authors

Faina M. Zakharova

Institute of Experimental Medicine; Saint Petersburg State University

Author for correspondence.
Email: fzakharova@mail.ru
ORCID iD: 0000-0002-9558-3979
SPIN-code: 9699-5744

Cand. Sci. (Biology), Senior Research Associate at the Department of Molecular Genetics, Senior Lecturer at the Department of Embryology

 

Russian Federation, Saint Petersburg; Saint Petersburg

Nadezhda A. Yagovkina

Saint Petersburg State University

Email: st110082@student.spbu.ru
ORCID iD: 0009-0002-3090-9621

2nd year graduate student of Faculty of Biology, Departments of Embryology

Russian Federation, Saint Petersburg

Vladislav V. Zakharov

Institute of Macromolecular Compounds of the Russian Academy of Sciences

Email: vlad.v.zakharov@mail.ru
ORCID iD: 0000-0002-7871-632X
SPIN-code: 1203-0639

Cand. Sci. (Biology), Research Associate at the Laboratory No. 5 (natural polymers)

Russian Federation, Saint Petersburg

References

Supplementary files