Development 100, 713-722 (1987) 713 Printed in Great Britain © The Company of Biologists Limited 1987

Spreading of microinjected horseradish peroxidase to nondescendant cells in embryos of (, )

WIEL M. KUHTREIBER, FLORENCI SERRAS and JO A. M. van den BIGGELAAR

Department of Experimental Zoology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands

Summary

We have injected horseradish peroxidase (HRP) and Injections of HRP into the blastocoelic cavity gave fluorescein-isothiocyanate dextran (FD) into cells and essentially the same results. In these cases, the HRP into the blastocoelic cavity of Patella vulgata em- was taken up by the cells from contact stage onwards. bryos, before and during the interval between 5th and When FD was injected into the blastocoelic cavity, no 6th cleavage, in which the mesodermal stem cell is uptake was observed, not even after prolonged pres- determined by means of interactions between the ence of FD in it. However, when HRP and FD were central 3D macromere and the contacting mixed, both were taken up, starting at contact stage. micromeres. Differences in labelling pattern of HRP, as compared Intracellular injections of HRP at different stages with FD, and a shift of the FD fluorescence after showed that, whereas before this contact phase no uptake, suggest that receptor-mediated endocytosis is spreading of label was observed, a clear intercellular involved. The possible morphogenetic significance of transfer of HRP was found after the contact was the transfer mechanism is discussed. established. Control experiments showed that it was Key words: intercellular communication, horseradish HRP in its intact, high molecular weight form that peroxidase, endocytosis, Patella, HRP, was transferred in the living embryo. fluorescein-isothiocyanate dextran.

Introduction does not appear to play a role, as in this case more than one macromere would be induced. This, how- In embryos of equal cleaving molluscs like Patella, the ever, does not occur (van den Biggelaar, 1977; van mesodermal stem cell is determined during the inter- den Biggelaar & Guerrier, 1979). The second and val between 5th and 6th cleavage. Out of four third possibility may be of importance in the Patella possible candidates (i.e. the four macromeres), only embryo. the macromere that contacts the animal micromeres In Patella embryos, gap junctions are present from will be induced (van den Biggelaar, 1977; van den the 4-cell stage onwards (Dorresteijn, Bilinski, van Biggelaar & Guerrier, 1979; van den Biggelaar, den Biggelaar & Bluemink, 1982). However, no dye Dorresteijn, de Laat & Bluemink, 1981). The mech- coupling is found until the 32-cell stage (Dorresteijn, anisms of the cellular interactions involved in this Wagemaker, de Laat & van den Biggelaar, 1983). inductive process during the contact phase are still Although, after the 5th cleavage, all cells bordering unknown. each other along the periphery of the embryo are dye Three main mechanisms are supposed to be in- coupled, no direct dye coupling between the pre- volved in induction in general (Slack, 1983): (1) sumptive mesodermal stem cell (3D) and the over- exchange of diffusible molecules over relatively long lying micromeres could be observed. From these distances, (2) exchange of signal molecules via inter- experiments on the spreading of low molecular cellular communication channels (Hooper & Subak- weight dyes, no definite conclusions could be drawn Sharpe, 1981; Finbow, 1982; Caveney, 1985) and (3) about the type of communication channel by which direct or indirect cell-to-cell contact for ligand-recep- they were transferred. The most obvious candidates tor coupling (Kiihtreiber et al. 1986). Exchange of are gap junctions, which allow the passage of mol- diffusible molecules over a relatively long distance ecules up to a relative molecular mass (Mr) of about 714 W. M. Kuhtreiber, F. Senas and J. A. M. van den Biggelaar

1200 (Simpson, Rose & Loewenstein, 1977). In order pressure system, which is described in detail elsewhere to obtain additional information about intercellular (Kuhtreiber & Serras, 1987). channels and other possible transfer mechanisms that For intracellular injections, micropipettes with outer tip may exist in a 32-cell stage Patella embryo, we diameters smaller than 0-1 urn (i.e. below the resolving decided to label cells with the enzyme horseradish power of the light microscope) were used. The tracers 3 (HRP, FD or mixtures of HRP and FD) were dissolved in peroxidase (HRP, M approx. 40X10 ). t 0-05 M-KCI or bidistilled water. Both solvents gave the same HRP can be easily detected and has been used as a results and the embryos developed normally. The injected tracer in a number of cell lineage studies in different amounts varied between 0-5 and 2pi, as measured by developing systems (Weisblat, Sawyer & Stent, 1978; performing comparable injections into paraffin oil and Nishida & Satoh, 1983; Goodall & Johnson, 1984; determining the volume of the liquid droplet. Weisblat & Blair, 1984; Weisblat, Kim & Stent, 1984; Injections into the blastocoelic cavity were performed Cruz & Pederson, 1985; Nishida & Satoh, 1985). In with micropipettes having outer tip diameters of 0-5 jim, these studies, HRP did not spread from the labelled which were firepolished by the puller. The tip was inserted cell to other blastomeres. During the successive between two lateral micromeres and advanced into the stages, it could therefore only be detected in the blastocoelic cavity. The injections were performed from progeny of the impaled cell. However, in our exper- immediately before to approx. 20 min after the completion of cytokinesis of 5th cleavage. At these stages, the embryos iments, the HRP did spread from impaled cells to have a large blastocoelic cavity and the cells are not other blastomeres. Therefore, we have to assume the flattened against each other, which makes it easier to presence of an additional mechanism, besides gap penetrate between cells. The injected amounts varied from junctions, for intercellular transfer in 32-cell-stage 5 to 10pi. Control injections with 005M-KC1 have shown Patella embryos. that development remains normal after blastocoelic injec- tions of up to 40 pi. Materials and methods Processing of embryos Embryos Embryos injected with FD were examined in vivo with a fluorescence microscope. Embryos injected with HRP were Adult specimens of the marine gastropods Patella vulgata fixed in either 4% formaldehyde in PBS (01 M, pH7-4) or and P. coerulea were collected in Roscoff or Dieppe 2% glutaraldehyde in PBS during at least 15 min. Control (France) and Arenys de Mar (Spain), respectively. They experiments have shown that endogenous peroxidase is were kept at 15°C in tanks with recirculating filtered inactivated during this fixation period. Next, the embryos seawater. Artificial fertilization was performed as described were washed in PBS and stained for 10 min with a mixture by van den Biggelaar (1977). Embryos of the appropriate of 1 nig ml"1 3,3'-diaminobenzidin (DAB, Sigma Chemical stage were selected. To facilitate the intracellular injec- Company) and 001% H2O2 in 0-lM-Tris buffer, pH7-6, tions, the embryos were washed in acidified seawater according to Graham & Karnovsky (1966). Proper inacti- (pH4-0) for 2-3 min to remove the gelatinous egg capsule vation of endogenous peroxidase was monitored by means which develops after the chorion has been stripped off. of several uninjected control embryos which were pro- Embryos treated in this way continued to develop normally. cessed in the same way as the injected ones. These control All subsequent injection experiments were carried out at embryos remained completely unstained. After dehy- 18°C in Millipore-filtered seawater. dration, clearing in xylene and embedding in Canada Balsam, the embryos were observed in a Zeiss light Microinjections microscope, either using brightfield or darkfield optics, or (1) Iontophoresis Nomarski differential interference contrast for making Microelectrodes (tip diameter smaller than 0-1 ^m, tip optical sections. resistance <100MQ when filled with 3M-KC1) were pulled from glass capillaries with inner filament (Clark Electro- Electrophoresis medical Instruments GC150F-15), and back-filled with a To assess the purity of the HRP preparations that have 3% solution of HRP (type II or VI, Sigma Chemical been used, they were analysed under nondissociating con- 3 Company) or FITC-dextran (FD, Mr 41xlO , Sigma) in ditions in a discontinuous Tris/glycine sodium dodecyl 0-IM-KCI. Iontophoresis was carried out essentially as sulphate electrophoresis system as described by Laemmli described by Dorresteijn et al. (1983) with some modifi- (1970) using a 4-5-26-8 % gradient running gel and a 4 % cations. HRP or FD was introduced into a cell using stacking gel, in a Biorad slab gel apparatus. Stock solutions repetitive depolarizing pulses of 10 nA amplitude and 0-4 s were prepared according to Lugtenberg et al. (1975) and the duration at intervals of 3-4s for about 7 min, or, alterna- gradient gel according to Tuszinski, Buck & Warren (1979). tively, using four or five long pulses of 8-9 nA amplitude for Acrylamide was purchased from Serva and recrystallized about 10 s each at intervals of 10-20 s. according to Loenig (1967). Methylene bisacrylamide and a molecular weight standard mixture were purchased from (2) Pressure injections Biorad. 10 or 20^1 samples of HRP (lmgrnP1), MP II 1 Direct (pressure) injections into cells or into the blasto- microperoxidase (lmgml" , Sigma Chemical Company) coelic cavity were performed with a thermal expansion and the molecular weight standard mixture were loaded on Spreading of HRP in Patella embryos 715 duplicate wells. Electrophoresis was carried out at 25 mA micromeres and the presumptive mesodermal stem constant current. Different gels were stopped at varying cell (3D), our investigation was focused on the times before or after the ionic front leaves the gel. This spreading of label between these two cell types. procedure ensured that no low molecular weight com- Although all four macromeres of a 32-cell-stage ponents were missed, either because they had already left embryo have the capacity to give rise'to the mesoder- the gel, or because they, were not yet completely focused. One part of the gel was stained in Coomassie Brilliant Blue mal stem cell (van den Biggelaar & Guerrier, 1979), R250 (0-25 % in 50 % methanol and 10 % acetic acid in aqua usually one of the two vegetal cross-furrow macro- bidest) and the other part was washed twice for 5min in meres will actually do so, because these have a PBS and then stained in DAB/H2O2 for 15min. Longer slightly advantageous spatial position within the em- periods did not result in a more intense staining, even when bryo as compared to the two other macromeres. If staining was performed overnight. The staining reaction one of these cross-furrow macromeres is injected, was stopped by washing in PBS and the gel was fixed in there will be a probability of approx. 50 % that this methanol/acetic acid. will turn out to become 3D. At later stages (at V + 50-60, i.e. when contact with the overlying Results animal micromeres is established), 3D has attained a central position in the embryo and can be readily Embryos injected by iontophoresis or by pressure identified. The results described below represent a continued to develop normally and in step with summary of a total of more than 200 successful control embryos, so we concluded that neither the impalements, injection procedure nor the injected substances were Single blastomeres of embryos at stages varying harmful. The results achieved with pressure injec- from the 2-cell to the early 32-cell stage were injected tions and with iontophoresis were essentially the with tracers (HRP II, HRP VI, FD). Both types of same. This indicates that electrical stimulation of the HRP gave essentially the same results, but as the impaled cell during iontophoresis did not influence specific activity of HRP type VI is much higher than the results. Also, there was no visible difference in that of HRP type II, the clearest results were results or embryonic development, whether the obtained with the former. When injected embryos pressure injections were performed with the tracers were investigated before V + 50-60, labelling was dissolved in aqua bidest or in 0-05M-KC1. In the restricted either to the impaled cell or to its offspring following, an embryo at stage x min after the com- in cases where one or more cleavages were allowed pletion of cytokinesis of 5th cleavage will be referred between injection and tracer visualization (Fig. 1). to as an embryo at stage V + x. However, an unmistakable spreading of HRP-label was observed when fixation was performed at V + Intracellular injections 50-60 or later. As we were especially interested in the communi- When HRP was injected into the future 3D macro- cation between the mesoderm-inducing animal mere, and fixation and staining of the embryo was

B C — Fig. 1. Intracellular injections of HRP in P. vulgata embryos. The embryos were fixed before the transfer of label started. (A) Animal pole view of an embryo injected at 4-cell stage and fixed at 8-cell stage. Only the two daughter cells of the injected cell are labelled (arrows). Darkfield optics. (B). Embryo injected at 4-cell stage and fixed at 16-cell stage. Only the four progeny cells of the injected cell are labelled. Note the spiral cleavage pattern that is typical for equally cleaving molluscs. Brightfield optics. (C) Optical section of an embryo of which the central 3D macromere was injected at V + 30 and fixed at V + 50. The embryo was fixed just before the transfer of HRP would start. Note the strong labelling of the nucleus. Nomarski optics. Bar, 25 [im. 716 W. M. Kiihtreiber, F. Serras and J. A. M. van den Biggelaar performed at V + 50-60 or later, the overlying animal distribution of HRP over the blastomeres is preceded micromeres were stained, as well as the blastomeres by an intermediate phase in which the animal and surrounding 3D at the vegetal pole (Fig. 2). The vegetal cells are more heavily labelled than the spreading of stain was fast: within a few minutes after equatorial cells interposed between them (Fig. 2A). an injection, the first nondescendant blastomeres When the macromeres 3A, 3B or 3C were injected, were positively labelled. When an embryo was fixed spreading of label from the impaled cell to surround- this soon after injection, apart from the injected 3D ing cells was also found, starting at the contact stage cell, only a few cells in the animal pole region were (V + 50-60). Spreading was found even when only a stained. When more time elapsed between injection very small amount of HRP was injected. Fig. 2B and fixation, more cells were labelled (Fig. 2C). shows such an injection into one of the most animal The possibility cannot be excluded that the animal micromeres. Note that in these cases HRP was first micromeres received the label indirectly from the detected in the nuclei (Fig. 2B). vegetally and meridionally located cells rather than As HRP was detected by means of DAB/H2O2 directly from 3D. This is unlikely, however, as staining after fixation of the injected embryo, it could

Fig. 2. Intracellular injection of HRP in Patella embryos. The embryos were fixed after the transfer of label started. (A) Brightfield optical section of a P. vulgata embryo injected with HRP VI at V + 10 in one of the two cross-furrow macromeres. The injected macromore turned out to be the central 3D macromere in this experiment. The embryo was fixed at V + 60. Note that the equatorial cells are less labelled than the injected 3D macromere and the overlying micromeres. (B) Animal pole view of P. vulgata embryo injected with HRP II in one of the most animally located micromeres at stage V + 45. The embryo was fixed at V + 50. Only some animal micromeres appear to be labelled. Note the strong labelling of the nuclei. Darkfield optics. (C) P. coerulea embryo injected with HRP VI at V + 20 in one of the vegetal cross-furrow macromeres and fixed at V + 75. Most cells are stained. The differential staining pattern as represented by this embryo is more clear in P. coerulea than it is in P. vulgata. Brightfield optics. (D) P. vulgata embryo injected with HRP VI at V + 70 in 3D (arrow) and bisected at V + 75, prior to fixation. Both halves contain HRP- positive cells. The asterisk indicates an uninjected control embryo. Darkfield optics. Bars: A,B,C, 25 fim; D, 50jum. Spreading of HRP in Patella embryos 111 not be excluded that HRP might diffuse aspecifically cavity was higher than the amount injected into a cell. during fixation or that the staining product might Shortly before the contact stage was reached, the diffuse. Therefore, impaled embryos were divided injected HRP accumulated near the membranes into animal and vegetal halves prior to fixation by use facing the blastocoelic cavity. It was not possible to of a glass needle. After such an experiment, cells in establish from optical sections whether the HRP both halves appeared to be stained, indicating that it accumulated inside or outside the cells (Fig. 3B). If it must have been the HRP in its intact form that spread was inside the cells, this might indicate that the first in the living embryo (Fig. 2D). uptake started shortly before the contact between 3D and the micromeres was established. Blastocoelic cavity injections From the above results, we could not exclude the Although, at this stage of the investigation, it is not possibility that the observed spreading of label after yet clear which mechanism is responsible for the injection of HRP into the blastocoelic cavity might be transfer phenomenon described above, it seems likely due to the movement of label during or after fixation. that the uptake of label in the recipient cells may be a Therefore, we injected FD into the cleavage cavity at process related to endocytosis. To investigate this various stages after 5th cleavage and observed the possibility, we decided to inject tracers into the injected embryos in vivo. We never observed uptake blastocoelic cavity, although the mechanism(s) for of FD from the blastocoelic cavity, not even after the the uptake of substances from the blastocoelic cavity prolonged presence of FD (Fig. 3C). The cells of the may not necessarily be identical to the mechanism(s) injected embryos seemed to show a diffuse labelling, that cause intercellular transfer. but from experience we know that this type of image When HRP was injected into the blastocoelic is due to internal reflections, in this case caused by the cavity at late 4th cleavage stages or at early stages FD in (remainders of) the blastocoelic cavity. When after 5th cleavage, and fixation and staining of the Fig. 5B is compared to Fig. 3C, it is obvious that embryo were performed before it had reached stage massive uptake of FD looks different from the result V + 50-60, the label was found only in the blasto- represented in Fig. 3C. coelic cavity and not in the cells, except occasionally The difference in behaviour of HRP and of FD in a cell that was damaged, but not killed, during the after blastocoelic cavity injections again raised the injection (Fig. 3A). When processing of an injected question whether the spreading of HRP was due to embryo was performed later than V + 60, many cells aspecific diffusion during or after fixation, or whether were positively labelled, often in a very regular real intercellular transfer occurred. Therefore, we pattern (Fig. 4). Thus, these results were analogous decided to inject a mixture of HRP and FD (see to the results obtained with intracellular injections of Fig. 5). Whereas before the contact stage neither HRP as far as the start of label spreading was HRP nor FD was internalized (Fig. 5A), both were concerned. After injection in the blastocoelic cavity, taken up at stages later than approx. V + 60 however, the results were clearer, probably because (Fig. 5B). A slight shift towards yellower wave- the amount of HRP injected into the blastocoelic lengths of the FITC fluorescence was visible after the

3A

Fig. 3. Blastocoelic cavity injections of HRP and of FD in P. vulgata embryos. The embryos were fixed before the start of uptake. (A) Nomarski optical section of an embryo injected with HRP VI at V + 10. The embryo was fixed at V + 40. HRP is only present in the blastocoelic cavity. (B) Nomarski optical section of an embryo injected at V + 10 and fixed at V + 50. The cell indicated by the arrow was probably damaged, but not killed, during the injection. (C) Fluorescence photomicrograph of living embryos injected with FD only at V + 20. The micrograph was taken at V + 95. The asterisk indicates an uninjected control embryo. Due to internal reflections, the cells seem to be positively labelled. The arrow indicates a cell that was damaged during the injection. Bars: A,B, 25 ^im; C, 50 jim. 718 W. M. Kuhtreiber, F. Serras and J. A. M. van den Biggelaar

Fig. 4. Blastocoelic cavity injections of HRP VI in 32-cell stage P. vulgata embryos. The embryos were fixed after the start of uptake. (A) Embryo injected at V + 5 and fixed at V + 50. The HRP has been taken up by several micromeres. Darkfield optics. (B) Animal pole view of an embryo injected at V + 30 and fixed at V + 60. Only the second quartet micromeres are unlabelled. The arrow indicates the polar bodies. Darkfield optics. (C) Vegetal pole view of the embryo shown in B. Also the four macromeres are positively labelled. This embryo was impaled between the two cross-furrow macromeres. The arrow indicates HRP'that leaked out during the injection. Darkfield optics. Bars, 25 fim.

FD was internalized by the cells. This was not quantified, but only assessed by visual examination. The whole embryo was FD labelled, including the cells that remained unstained when tested for the presence of HRP (cf. Fig. 4). In contrast, no uptake of FD in any cell was observed when it was injected alone. Another difference between the behaviour of FD and HRP was that at very late 5th cleavage stages (i.e. V + 90-100 or later), the fluorescence of FD was still present, whereas no HRP was detectable any more, except for HRP inside a cell that was damaged during the injection.

Electrophoresis Before one can conclude that the 32-cell embryo of Patella must have an intercellular transfer mechanism for HRP, one has to be sure that the HRP prep- arations do not contain stainable degradation prod- ucts small enough to pass through gap junctions. DAB/H2O2 and Coomassie Brilliant Blue staining of HRP preparations on SDS-polyacrylamide gels did not reveal low molecular weight components, even though the amount of HRP loaded on a well was in the order of 5xl06-fold in excess of the estimated amount injected into a cell (Fig. 6).

Fig. 5. Fluorescence photomicrographs of living P. Discussion vulgata embryos injected in the blastocoelic cavity with a mixture of HRP VI and FD. (A) Embryo injected at V + 10 and photographed at V + 30. A large blastocoelic The results described in this paper clearly show that cavity is still present. The cells are still unlabelled at this HRP is not a reliable cell lineage tracer in each stage. (B) Embryos injected at V + 20 and photographed developmental system. In Patella, HRP is transferred at V + 75. The FD is now taken up by the cells. from a well-defined stage onwards. The time at which Uninjected control embryos are indicated by asterisks. the observed phenomenon starts is precisely deter- Bars, 25 fim. mined at V + 50-60. Such a consistent timing in an Spreading of HRP in Patella embryos 719 embryonal system argues in favour of a physiologi- the observed spreading reflects a mechanism that cally and developmentally significant communication might play an important role in the interaction event. The start of tracer spreading coincides with, or between 3D and the micromeres, leading to the only slightly precedes, the contact between the cen- induction of the mesodermal cell line. tral macromere 3D and the overlying micromeres. Before discussing the possible nature of the trans- Therefore, the possibility should be considered that fer mechanism(s) involved, we will first evaluate the question whether the observed spreading might be 12 3 4 5 1 2 due to an artefact. First, the strongest argument against artefact comes from the experiments in which a HRP-injected embryo is bisected before fixation. The results of these experiments indicate that inter- cellular transfer of HRP has occurred in the living embryo. Second, the moment at which transfer of -3 X10 HRP can first be observed is reproducible and phase specific. Third, it has been found that aspecific 92-5- spreading of HRP can result from overloading axons in axophoresis experiments (Brown & Fyffe, 1984). 66-2- However, this cannot be the case in our experiments, as spreading of label is also observed when only a small amount of HRP is injected (Fig. 2B). More- over, it would be difficult to explain why overloading 45 - in Patella should occur at V + 50 or later and not before that stage, and why the nuclei are labelled first. Fourth, Bennett (1973) has shown in Fundulus that nonspecific spreading of HRP after fixation will occur when fixation is performed in the presence of 31 - La(OH)3, but not when normal fixation procedures are used. The question then arises whether thie transfer of native HRP molecules accounts for the intercellular 21-5- spreading of label or whether this is the result of transfer of low molecular weight degradation prod- ucts through gap junctions. From the absence of low 14-4- molecular weight subunits of HRP on SDS-poly- acrylamide gels, we conclude that HRP is transferred in its native, high molecular weight form, although it cannot be excluded that HRP is degraded intracellu- larly, perhaps into components small enough to pass gap junctions. The latter assumption, however, is 1-9- very unlikely, as it would be difficult to explain why this would not happen at earlier stages. Dorresteijn et al. (1983) have shown that spreading of Lucifer Yellow in Patella starts at V +10-20. Moreover, B embryos that have been injected intracellularly with HRP at stages V + 50 or later, and fixed immediately Fig. 6. Gradient polyacrylamide gel electrophoresis of after completion of the injection, already show a HRP samples did not reveal low molecular weight considerable amount of spreading of DAB/H2O2- components small enough to pass through gap junctions. positive material. Extensive intracellular degradation (A) Coomassie Brilliant Blue staining. 1, microperoxidase of HRP in such a short time is highly unlikely. 1 MPII (lO/iglO/il" ); 2, Biorad low molecular weight Therefore, we may conclude that it is the intact HRP standard mixture (lysozyme, soy bean trypsin inhibitor, molecule that is transferred intercellularly. carbonic anhydrase, ovalbumin, bovine serum albumin and phosphorylase B); 3, HRP type II (lO/iglO^r1); Three generally accepted mechanisms for the inter- 4, HRP type VI (lO^glO^r1); 5, microperoxidase MPII cellular spreading of HRP can be envisaged: (1) (20/iglOjun1). Note that the microperoxidase is not passage through midbodies, as suggested by Goodall stained. (B) DAB/H2O2 staining. 1, microperoxidase & Johnson (1984); (2) passage through large inter- ); 2, HRP type VI ' cellular channels, e.g. in the form of cytoplasmic 720 W. M. Kiihtreiber, F. Serras and J. A. M. van den Biggelaar bridges as found in the embryo (Cartwright & uptake of FD would be much lower and thus not Arnold, 1980); Ginzberg, Morales, Spray & Bennett, detectable. This might also explain why Dorresteijn et 1985) and in the zebrafish embryo (Kimmel & Law, al. (1983) could not detect the direct transfer of 1985) or (3) exocytosis followed by endocytosis as Lucifer Yellow between the central 3D macromere found in synapses (Hongo etal. 1981; Triller & Korn, and the contacting micromeres. The mechanism pro- 1981). posed above can explain how HRP gets into the cells. Transfer via midbodies is very unlikely, as there is However, it does not explain how it gets out of them. no reason why they would not permit transfer before To explain the rate of endocytosis, one has to assume stage V + 50. The presence of intercellular channels that exocytosis is also stimulated. This remains to be large enough for HRP to pass through cannot be explained. excluded, although in previous electron microscopi- Summarizing, we may conclude first, that embryos cal studies no indications have been found for cyto- of Patella vulgata and P. coerulea have a mechanism plasmic bridges (Dorresteijn et al. 1982). The third for the transfer of specific substances at the 32-cell possibility, exocytosis of HRP followed by endo- stage. This mechanism may be stimulated by HRP cytosis by adjacent cells seems to be a likely one. If and must be different from junctional channels. endocytosis occurs, it is to be expected that the Further experiments are in progress to elucidate the HRP/FD mixture will be taken up into lysosomes. nature of this mechanism. Second, at least in these The low pH of the lysosomal fluid would account for molluscan embryos, HRP cannot be used as a reliable the observed shift in fluorescence of FD towards marker in cell lineage studies. longer wavelengths (Martin & Lindqvist, 1975; Gei- sow, 1984). Both FD and HRP would be digested in The authors wish to thank H. Goedemans, D. Schaap the lysosome, rendering the HRP inactive and liber- and H. Smits for their assistance. Prof. Dr N. H. Verdonk ating FITC, which is small enough to pass through and Dr M. R. Dohmen are thanked for reading the gap junctions. The free FITC could therefore spread manuscript critically and for their stimulating suggestions. throughout the whole embryo. This is in accordance The Department of Image-processing and Design is thanked for the photographic services rendered. This re- with the observation that at later stages the DAB/ search was supported by grants from the Foundation for H2O2 reaction for HRP is negative, whereas fluor- Fundamental Biological Research (BION), which is subsi- escence by FITC is still present. dized by the Netherlands Organization for the Advance- Another factor to be considered is that HRP and ment of Pure Research (ZWO), to W. Kiihtreiber; and FD are taken up by the micromeres, in spite of the from the Commission for the Advancement of Research fact that these cells are covered with a layer of and Technology (CIRIT), which is subsidized by the extracellular matrix shortly before and during the Generalitat de Catalunya, Spain, to F. Serras. contact phase (Kiihtreiber etal. 1986). HRP has been shown to be able to pass through such a layer. Modespacher, Rudin, Jenni & Hecker (1986) have References shown that HRP can pass the basal lamina of the midgut of the tsetse fly and is taken up into fatbody BENNETT, M. V. L. (1973). Permeability and structure of cells. Straus (1981, 1983a,/?) has shown that several electrotonic junctions and intercellular movements of cell types of the rat have mannose-specific binding tracers. In Intracellular Staining in Naerobiology (ed. S. sites for HRP. The binding of HRP to these receptors B. Kater & C. Nicholson), pp. 115-135. Berlin, Heidelberg, New York: Springer-Verlag. can be suppressed by competition with mannose or BROWN, A. G. & FYFFE, R. E. W. (1984). Intracellular glycoproteins. As the ECM of Patella micromeres staining of mammalian neurones. In Biological reacts positively with mannose-specific lectins and Techniques Series, pp. 1-115. New York: Academic contains glycoproteins (Kiihtreiber et al. 1986), simi- Press. lar binding sites may be present in a 32-cell-stage CARTWRIGHT, J. JR & ARNOLD, J. M. (1980). Intercellular Patella embryo. This implies that these embryos may bridges in the embryo of the Atlantic squid, Loligo possess an uptake mechanism for ECM, that enables pealei. I. Cytoplasmic continuity and tissue HRP to utilize a receptor-mediated endocytosis differentiation. J. Embryol. exp. Morph. 57, 3-24. mechanism to enter the cells by binding to the CAVENEY, S. (1985). The role of gap junctions in receptors normally utilized by ECM components. development. A. Rev. Physiol. 47, 319-335. CRUZ, Y. P. & PEDERSEN, R. A. (1985). Cell fate in the Our results suggest that HRP stimulates the endo- polar trochectoderm of mouse blastocysts as studied by cytosis process that would normally occur at a lower microinjection of cell lineage tracers. Devi Biol. 112, level in uninjected or FD-injected embryos. In the 73-83. presence of HRP the stimulation of endocytosis DORRESTEIJN, A. W. C, BILINSKI, S. M., VAN DEN would facilitate the nonspecific entry of FD into the BIGGELAAR, J. A. M. & BLUEMINK, J. G. (1982). The cells, whereas in the absence of HRP the rate of presence of gap junctions during early Patella Spreading of HRP in Patella embryos 721

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