Proc. Nati. Acad. Sci. USA Vol. 91, pp. 5622-5626, June 1994 Developmental Biology Spatial distribution of two maternal messengers in during oogenesis and embryogenesis (/in situ hybridization/mRNA localIzation/cel surface proteins/proteilocalizain) M. Di CARLO*t, D. P. ROMANCINO*t, G. MONTANA*, AND G. GHERSI* *Istituto di Biologia dello Sviluppo, Consiglio Nazionale delle Ricerche, 90123 Palermo, Italy; and *Dipartimento di Biologia Cellulare e deilo Sviluppo, 90123 Palermo, Italy Communicated by A. A. Moscona, February 28, 1994 (receivedfor review October 22, 1993)

ABSTRACT We demonstrated that two mRNAs that are structures; while observation indicated the presence of some synthesized during the vitellogenic period ofoogenesis and that asymmetry along the /vegetal axis of the unfertilized code for cell surface proteins are asymmetrically distributed in sea urchin egg, no satisfactory explanation for this asymme- the unfertilized egg of Paracentrotus lividus. At fertilization, try was provided, except for the suggestion of a double these RNAs rapidly localize in the cortical zone at the animal gradient proposed in 1928 by Runnstrom (10, 11). There was pole of the egg. They are then detected in the mesomeres and no evidence of a differential RNA localization in the macromeres, but not in the micromeres, and thereafter are eggs, as has been demonstrated in Xenopus and Drosophila found in the ectoderm but not in the vegetal plate, mesenchyme eggs. cells, or early intestine. They disappear in late gastrula. The We report here that two maternal mRNAs, bepi and bep4, proteins synthesized by these mRNAs show the same territorial both of 1.4 kb, are asymmetrically distributed in the unfer- location during the period examined here, which included the tilized egg of Paracentrotus lividus. They belong to a multi- unfertilized egg and the 16-blastomere stage. These conclusions gene family and code forproteins that become localized in the were reached on the basis of in situ hybridization and immu- cell surface during early embryogenesis and may be relevant ntaning experiments, as well as Northern and Western blot for cell adhesion (12, 13). Their molecular mass is 33 kDa, and analyses of isolated blastomeres. The possible sinificance of their messengers show a particular structural organization; this asymmetric distribution of these two mRNAs and proteins two well-conserved domains surrounding a single specific in the establishment of the animal/vegetal axis is discussed. domain (12, 13). We report here that both these mRNAs and the corresponding proteins are located exclusively in one-half Determination of the embryonic axes has long been a central of the unfertilized egg (probably the animal half) and become problem in developmental biology. Recent studies in Dro- localized at the animal pole immediately after fertilization. sophila on the underlying molecular mechanisms have dem- Those parts of the embryo that are derived from the vegetal onstrated that information that specifies the anteroposterior half of the egg are devoid of these two mRNAs. After the and the dorsoventral axes of the egg is already laid down gastrula stage, these mRNAs are no longer found in the during oogenesis (1) and that a cascade of events determines embryo. the position of the head and of the thorax. Certain maternal genes lead to localization of the product of the bicoid gene, MATERIALS AND METHODS a transcription factor, producing an anterior/posterior con- centration gradient that regulates zygotic target genes (2, 3). In Vitro Transcription Using pGEM Plsmid. The Sac I/Stu A maternal RNA, nanos, the product of which prevents the I and Ava I/Stu I fragments of bepi and bep4, respectively, translation of a transcription factor encoded by a ubiquitous containing specific parts of both clones (12), were subcloned maternal RNA is initially localized at the posterior pole (4). in the Sac I/HinclI and Ava I/HincII sites of the transcrip- Another cascade of events occurs in the establishment ofthe tion vector pGEM2. These constructs were used to synthe- dorsoventral axis in which the product of the maternal Toll size antisense RNA probes with a Riboprobe kit (Promega), gene, probably a membrane receptor, and the maternal dorsal according to the manufacturer's instructions with uridine gene are involved (5, 6). The possibility that determination of 5'-[a-[35S]thioltriphosphate (1000 Ci/mmol; 1 Ci = 37 GBq; the embryonic axes is correlated with different locations of Amersham). Probes were synthesized to a specific activity of maternal RNAs in different egg territories has also been 2 x 108 cpm/mg. The final concentration of the probe in the suggested for the eggs ofXenopus, where Melton (7, 8) found hybridization solution was equivalent to 12.5 x 106 cpm/ml. that the Vgl RNA, whose product shares homologies with a The presence of transcripts was analyzed by 6% polyacryl- type (3 transforming growth factor, is transferred to the amide/7 M urea gel. vegetal pole of the oocyte at the time of its maturation. In Situ Hybridization. Paraffin sections of eggs and em- In contrast to Drosophila and Xenopus, nothing is known bryos were spread onto polylysine-coated slides, as de- to date about the molecular basis ofaxis determination in sea scribed by Cox et al. (14). Hybridization and washing were urchin embryos, where the problem of an underlying mech- performed according to Cox et al. (14); the only modification anism was first raised at the beginning of this century. was inclusion of 10 mM 2-mercaptoethanol in all the washing Microsurgery experiments performed mostly by Horstadius steps. Slides were processed for autoradiography as de- (9) had demonstrated that if unfertilized eggs were equato- scribed by Cox et al. (14). Exposure time was 2 days for the rially cut into two halves and each half was fertilized, the sections shown. animal halves always developed into ciliated hollow spheres Oocyte Purification. Purification was carried out according ("animalized" embryos), whereas the vegetal halves pro- to Giudice et al. (15). Sea urchin female gonads were sus- duced "vegetalized" embryos with an excess of vegetal pended in 100 ml of Ca2+-free seawater containing 100 mg of Pronase (Calbiochem; B-grade) and the released oocytes The publication costs of this article were defrayed in part by page charge were freed from the gonads by filtration. The sedimented payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

5622 Downloaded by guest on October 1, 2021 Developmental Biology: Di Carlo et A Proc. Natl. Acad. Sci. USA 91 (1994) 5623 oocytes were layered on top of a 5-20% Ficoll gradient in RESULTS seawater and centrifuged in a swing-out rotor at 700 x g for We have previously demonstrated that two maternal 20 sec.Wehvprvosydmntae thttomenl Separation of Meso- and Macromeres and Micromeres. The andmRNAs,persistbepithroughoutand bep4,earlyaredevelopmenttranscribed upduringto theoogenesisgastrula cells were separated by a modification of the Harkey and stage, when they disappear (12). To investigate possible Whiteley procedure (16). Sea urchin eggs were fertilized in 2 changes in their territorial distribution within eggs and early mM p-aminobenzoic acid. Sixteen-cell stage embryos were embryos, we prepared two antisense RNAs, labeled as dissociated into single cells in Ca2+- and Mg2+-free seawater, described in Materials and Methods and shown in Fig. 1, and layered on top of a 5-20% Ficoll gradient in seawater and used them for in situ hybridization. centrifuged in a swing-out rotor at 1000 x g for 30 sec. Fig. 2A shows that both bepi (I) and bep4 (II) mRNAs are RNA Extraction and Northern Blot Hybridization. Total localized in only a part of the unfertilized egg, which corre- RNA was isolated by using an acid guanidinium isothiocya- sponds roughly to one-halfofthe egg. When the hybridization nate/phenol/chloroform method (17). The RNA (10-20 mg) was carried out on sections of embryos at the 16-cell stage was electrophoresed on 1% agarose/1.1 M formaldehyde gel (Fig. 2C), no hybndization was observed in the micromeres; and transferred onto nylon membranes (Amersham). Blots at later stages, there was no hybridization in the vegetal part were hybridized with labeled fragments specific for bepl and of the blastulae, in the primary mesenchyme cells, or in the bep4 (12) and with a fragment containing a portion of an 18S * h diztion was o i rRNA gene (gift from Rainer Barbieri) at 650C in 4x standard gut (Fig. 2 D and E), whereasbrhyobsrat all these stages This saline citrate/5x Denhardt's solution/0.5% SDS. strongly suggested that bepi and bep4 were confined to the Gel Electrophoresis and Western Blot Analysis. Samples (50 animal half of the unfertilized egg, unless one assumes that mg) were resuspended in an equal volume of 2x sample their localization became reversed in the interval between buffer (0.0625 M TrisHCl, pH 6.8/1% glycerol/0.2% SDS/ fertilization and the 16-blastomere stage. Of particular inter- 0.7 M 2-mercaptoethanol/0.1% bromophenol blue) and sub- est was the finding that after fertilization the two mRNAs jected to electrophoresis on SDS/10% polyacrylamide gel became localized close to the egg pole which is identified as (18). Proteins were then transferred onto nylon membranes the animal pole, for the reasons discussed above (Fig. 2B). (Amersham) using an LKB Multiphor blot apparatus. After Consistent with earlier evidence from Northern blots (12), in washing in Tris-buffered saline (TBS), filters were incubated situ hybridization was not observed in later stages (data not with primary antibodies, produced against a portion of bepl shown). and bep4 proteins, diluted 1:500 as described by Romancino The above evidence that micromeres did not contain bepi et al. (13). and bep4 RNAs was further confirmed by the following Immunostaining of Egg Sections. Paraffin sections of P. experiment: embryos at the 16-blastomere stage were disso- lividus eggs were prepared for staining according to Levi et ciated into cells, and the micromeres were separated from the al. (19). Sections (3 mm thick) were deparaffinized and mixture of mesomeres and macromeres by the procedure of incubated with the polyclonal antibodies anti-bepl and anti- Harkey and Whiteley (16) modified as shown schematically bep4 (13) diluted 1:100 in TBS according to a standard in Fig. 3A; total RNA was separately extracted from these procedure (20). After washing in TBS, the sections were two cell populations and the presence of bepi and bep4 was incubated with mouse anti-rabbit IgG peroxidase conjugate investigated by Northern blot analysis, using as probes (Promega) diluted 1:50 in TBS. After washing in TBS, the specific fragments ofboth cDNAs. As shown in Fig. 3 B and peroxidase color reaction was developed by bathing the C, in the micromeres there was no hybridization signal for sections in a solution containing diaminobenzidine (6 mg/ml), these two mRNAs, whereas a clear signal for both mRNAs 0.05 M Tris HCl (pH 7.6), and 0.03% H202. was obtained in the meso- and macromeres and in the 16-cell AL B

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FIG. 1. (A) Construction of clones for in vitro transcription. Zigzag and open square lines indicate the regions common to bepl and bep4. Hatched and dotted boxes indicate the specific region of bepi and bep4, respectively. Sac I, Ava I, Stu I, and Hincll show the restriction sites from which the fragments inserted into pGEM2 have been prepared. ORI, origin ofreplication; amp, ampicillin resistance site; Sp6 PR, promoter site for Sp6 polymerase. (B) Autoradiograms of fragments obtained by in vitro transcription; numbers of base pairs synthesized are indicated on the left. Downloaded by guest on October 1, 2021 5624 Developmental Biology: Di Carlo et al. Proc. Natl. Acad. Sci. USA 91 (1994)

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FIG. 3. Separation of meso- and macromeres and micromeres and Northern and Western blot analyses. (A) Schematic represen- tation of meso- and macromeres and micromere separation from 16-cell stage and RNA extraction from the corresponding fractions. CMFSW, Ca2+- and Mg2+-free seawater. (B and C) Northern blot of total RNA from entire 16-blastomere embryos (lanes 1), isolated micromeres (lanes 2), isolated meso- and macromeres (lanes 3), hybridized either with 18S rRNA (control), or with bepi (B) and bep4 (C) probes. Hybridization bands of each probe used are indicated by arrowheads. (D and E) Immunoblotting of proteins extracted from meso- and macromere (lanes 1) and micromere (lanes 2) fractions incubated with anti-bepl (D) and anti-bep4 (E) polyclonal antibodies. Molecular mass of the proteins is indicated on the left.

FIG. 2. Dark-field images ofthe location ofbepi and bep4 mKNA ies to these proteins (Fig. 4). Immunostaining with either by in situ hybridization on sections of unfertilized egg (A), fertilized anti-bepl (Fig. 4A) or anti-bep4 (Fig. 4B) antibody was egg (B), 16-cell stage (C), blastula (D), and gastrula (E) with detected only in that half of the egg identified as the animal single-strand antisense bepl (I) and bep4 (II) probes. half. At what stage ofoogenesis are these mRNAs synthesized? stage blastomere RNA. The presence of 18S which rRNA, To answer this question, we isolated oocytes from gonads was used as a evident in the micromere control, is, however, and separated them according to size in a Ficoll gradient by in same lane the figure. the method described by Giudice et al. (15). The RNAs were To investigate whether the proteins produced by the bepi separately extracted from the small oocytes ranging in diam- and bep4 RNAs were also localized in the same territories, eter from 30 to 50 pm and from larger ones ranging in we carried out a Western blot analysis of proteins extracted diameter from 50 to 100 ,um. Northern blot analysis (Fig. 5A) from isolated micromeres or from meso- and macromeres. As demonstrated that bepi and bep4 RNAs were not yet detect- shown in Fig. 3 D and E, the bepl (Fig. 3D) and bep4 (Fig. able in the small oocytes, although these oocytes hybridized 3E) proteins were found to have the same spatial distribution with the control probe for the 18S rRNA. On the other hand, as their corresponding RNAs. These results were confirmed clear hybridization bands were obtained with the larger by immunostaining unfertilized eggs with polyclonal antibod- oocytes, indicating that the synthesis ofbepi and bep4 RNAs Downloaded by guest on October 1, 2021 Developmental Biology: Di Carlo et al. Proc. Natl. Acad. Sci. USA 91 (1994) 5625 detected in the micromeres, vegetal plate, mesenchyme cells, B and early intestine. Also, the corresponding proteins show the same localization pattern in the unfertilized egg and at the 16-blastomere stage (where they have been studied so far). The synthesis of these mRNAs appears to start during oogenesis during the vitellogenic period. As mentioned in the Introduction, axis-determining substances were demon- strated in the Drosophila egg (1) to be the products of maternal genes and of a cascade of processes including cell surface receptors such as products ofthe genes torso (21) and Toll (5). The resulting chain of events culminates in the differential activation of zygotic genes that bring about the FIG. 4. Immunostaining of section of unfertilized eggs using formation of different structures in the embryo. polyclonal antibodies against bepl (A) and bep4 (B). We propose that a fundamentally similar chain of events occurs also in the sea urchin. It is known that certain genes commenced during the vitellogenic period. Differences in the are preferentially activated in different territories of the intensity of 18S rRNA hybridization between the small and embryo. The earliest genes whose activation was demon- the larger oocytes were due to different amounts of RNA (20 strated in animal blastomeres are the VEBs (22). Later, the and 10 mg, respectively) loaded on the gel. genes for the hatching enzyme and for a tolloid-like protein Are the bepl and bep4 RNAs synthesized directly in the are turned on (23, 24) and expressed only in the animal oocytes or in the follicular cells and then transferred into territories. The Spec genes follow (25) and are preferentially oocytes? The results of in situ hybridization performed on expressed in the aboral ectoderm cells and their precursors, entire ovaries using bepi as probe (Fig. SB) favor the first while the SM50 (26) and Endol (27) genes are expressed only possibility, because of the localization of these RNAs in the in the mesenchyme and endoderm, respectively. The CyIIIa oocyte nuclei-i.e., presumably at the site of their synthesis (28) gene also is expressed in the aboral ectoderm. Still, other (Fig. SBa). As expected from the experiments with isolated sea urchin genes have territorial expressions (for a review, oocytes, the radioactivity was observed mainly in the larger see ref. 29) that correspond to the specific morphogenesis of oocytes and, in some ofthese, it has already been transferred those territories. However, what is presently unknown is the almost completely from the nucleus into the cytoplasm (Fig. nature of the initial egg asymmetry that starts the cascade of SB b and c). In some of the larger oocytes, there appeared to gene activations leading to axis determination and morpho- be an asymmetric localization of these RNAs in the cyto- genesis. We hypothesize that the territorial location of the plasm (Fig. 5Bb). two maternal mRNAs and their products described here in one-halfofthe unfertilized egg represents the asymmetry that DISCUSSION starts such a cascade of gene activation. It is of potential interest that the proteins produced by these two maternally We found that in the egg of the sea urchin P. lividus there is derived mRNAs appear to be associated with the cell surface an asymmetric distribution oftwo mRNAs that correspond to (13). cell surface proteins even in the unfertilized egg. At fertili- Assuming that the territorial localization of bepi and bep4 zation, these mRNAs become localized in the cortical zone genes generates a localized expression of cell surface pro- at the egg pole identified as the animal pole. These mRNAs teins, it is conceivable that these proteins function in recep- are confined mostly to the animal parts of the embryo until tion of specific developmental signals derived, perhaps, from the gastrula stage, at which time they disappear. They are not the micromeres. Davidson (30, 31) suggested that mi-

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FIG. 5. (A) Temporal expression of bepl and bep4 mRNAs during oogenesis. Total RNA extracted from small (20 mg) (lanes 1) and large (10 mg) (lanes 2) oocytes hybridized either with 18S rRNA or with bepl and bep4 probes. Schematic drawing of oocytes of different sizes used is presented under the corresponding lanes. Hybridization bands of each probe are indicated by arrowheads. (B) Spatial expression of bepi mRNA during oogenesis. In situ hybridization using bepl probe reveals localization of the corresponding mRNA in the nuclei (a), in the nuclei and in the cytoplasm (b), and only in the cytoplasm (c) of some of the large oocytes. Downloaded by guest on October 1, 2021 5626 Developmental Biology: Di Carlo et al. Proc. Natl. Acad. Sci. USA 91 (1994) cromeres are responsible for a cascade of cell interaction- 4. Frohnhofer, H. G. & Nusslein-Volhard, C. (1986) Nature (Lon- activated events, leading to the early morphogenesis of the don) 324, 120-125. sea urchin embryo. That the bepl and bep4 proteins may 5. Hashimoto, C., Hudson, K. L. & Anderson, K. V. (1988) Cell in interactions was suggested by experiments 52, 269-279. have a role cell 6. Anderson, K. V., Jurgens, G. & Nusslein-Volhard, C. (1985) in which treatment of dissociated embryo cells with Fabs Cell 42, 779-789. against bepl and bep4 proteins caused inhibition of cell 7. Melton, D. A. (1987) Nature (London) 328, 80-83. reaggregation (13). On the other hand, these proteins may 8. Melton, D. A. (1991) Science 252, 234-241. have other roles because, as previously shown (13), they are 9. Horstadius, S. (1928) Acta Zool. 9, 1-4. found not only on the cell surface but also within the 10. Runnstrom, J. (1928) Wilhelm Roux' Arch. Entwicklungsmech. cytoplasm. Analysis of the primary structure of these pro- Org. 113, 556-581. teins, from the nucleotide sequence, has not revealed 11. Runnstrom, J. (1928) Acta Zool. 9, 365-423. inferred 12. Di Carlo, M., Montana, G. & Bonura, A. (1990) Mol. Rep. Dev. any resemblance to known proteins. Their amphipathic struc- 25, 28-36. ture, however, shows similarities to that of the gp8O cell 13. Romancino, D. P., Ghersi, G., Montana, G., Bonura, A., surface protein of Dictyostelium, which is known to have a Perriera, S. & Di Carlo, M. (1992) Differentiation 50, 67-74. role in cell adhesion during the aggregation stage (32). Ex- 14. Cox, K. H., DeLeon, D. V., Angerer, L. M. & Angerer, R. C. periments to verify the suggested role of the asymmetric (1984) Dev. Biol. 101, 485-502. distribution of these two mRNAs and proteins in embryonic 15. Giudice, G., Sconzo, G., Bono, A. & Albanese, I. (1972) Exp. axis determination by treatment of eggs with antibodies Cell Res. 72, 90-94. 16. Harkey, M. A. & Whiteley, A. H. (1980) Wilhelm Roux's Arch. against bepl and bep4 proteins remain to be done. Dev. Biol. 189, 111-122. The finding that after fertilization these mRNAs are de- 17. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, tected only in the cortical zone of the animal pole suggests W. J. (1979) Biochemistry 18, 5294-5299. movement ofthese mRNAs to that region. This suggestion is 18. Laemmli, U. K. (1970) Nature (London) 227, 680-685. supported by a count ofthe radioactive grains that showed a 19. Levi, G., Crossin, L. & Edelman, G. M. (1987) J. CellBiol. 105, 40%o increment ofgrain concentration in the egg cortex versus 2359-2371. the endoplasm after fertilization. Although other hypotheses 20. Harlow, E. & Lane, D. (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY). cannot be presently excluded, movement of the mRNAs 21. Sprenger, F., Stevens, L. & Nusslein-Volhard, C. (1989) Na- seems the most plausible possibility that is also consistent ture (London) 338, 478-483. with the translocation of Vgl mRNA to the vegetal pole 22. Reynolds, S. D., Angerer, L. M., Palis, J., Nasir, A. & An- during the maturation of Xenopus oocytes (7). Since this gerer, R. C. (1992) Development 114, 769-786. translocation apparently involves microtubules and microfil- 23. LePage, T., Sardet, C. & Gache, C. (1992) Dev. Biol. 150, aments (33, 34), whether these structures play a role in the 23-32. movement of bepi and bep4 RNAs to the animal pole of the 24. LePage, T., Ghiglione, C. & Gache, C. (1992) Development of their 114, 147-163. sea urchin egg and whether any part nucleotide 25. Hardin, P. E., Angerer, L. M., Hardin, S. H., Angerer, R. C. sequences is relevant to their translocation and anchoring & Klein, W. (1988) J. Mol. Biol. 202, 417-431. need to be investigated. 26. Benson, S., Sucov, H., Stephens, L., Davidson, E. & Wilt, F. (1987) Dev. Biol. 120, 499-506. We are deeply indebted to Prof. Giovanni Giudice for his contin- 27. Wessel, G. M. & McClay, D. R. (1985)Dev. Biol. 111, 451-463. uous and enthusiastic encouragement during this work and for help 28. Franks, R. R., Hough-Evans, B. R., Britten, R. J. & David- in manuscript preparation. We thank Dr. Roberto Gambino for his son, E. H. (1988) Genes Dev. 2, 1-12.28. continuous and helpful discussions and Dr. Rainer Barbieri for the 29. Giudice, G. (1993) Anim. Biol. 2, 67-73. gift of the 18S rRNA probe. 30. Davidson, E. H. (1990) Development 108, 365-389. 31. Ransick, A. L. & Davidson, E. H. (1993) Science 259, 1134- 1. St. Johnston, D. & Nusslein-Volhard, C. (1992) Cell 68, 201- 1138. 211. 32. Chi-Hung, S. (1990) BioEssays 12, 357-362. 2. Nusslein-Volhard, C., Frohnhofer, H. G. & Lehmann, R. 33. Yisraeli, J. K., Sokol, S. & Melton, D. A. (1989) Development (1987) Science 238, 1675-1681. 1989 Suppl., 31-36. 3. Driever, W. & Nusslein-Volhard, C. (1989) Nature (London) 34. Yisraeli, J. K., Sokol, S. & Melton, D. A. (1990) Development 337, 138-143. 108, 289-298. Downloaded by guest on October 1, 2021