Cell division and cell allocation in early mouse development

Transcription

1 /. Embryol. exp. Morph. Vol. 8, pp , Printed in Great Britain Company of Biologists Limited 1978 Cell division and cell allocation in early mouse development By S. J. KELLY, 1 J. G. MULNARD 2 AND C. F. GRAHAM, 13 From the Department of Zoology, Oxford SUMMARY Cell division was observed in intact and dissociated mouse embryos between the 2-cell stage and the blastocyst in embryos developing in culture. Division to the -cell stage was usually asynchronous. The first cell to divide to the -cell stage produced descendants which tended to divide ahead of those cells produced by its slow partner at all subsequent stages of development up to the blastocyte stage. The descendants of the first cell to divide to the -cell stage did not subsequently have short cell cycles. The first cell or last cell to divide from the -cell stage was labelled with tritiated thymidine. The embryo was reassembled, and it was found that the first pair of cells to reach the 8-cell stage contributed disproportionately more descendants to the ICM when compared with the last cell to divide to the 8-cell stage. INTRODUCTION The cells of the preimplantation mouse embryo do not divide in synchrony with each other and this raises the possibility that physiological differences exist between the cells of the embryo (e.g. Dalcq, 1957; Mulnard, 1967; Borghese & Cassini, 1963; Lewis & Wright, 1935). We have investigated division asynchrony and the relationship between this asynchrony and the allocation of cells to the inner cell mass (ICM) and to the trophectoderm of the blastocyst. MATERIALS AND METHODS Supply and culture of embryos The embryos were obtained from natural matings of a variety of strains which are indicated in the figure legends. The embryos were dissected into prewarmed, pre-equilibrated Whitten's (1971) medium and cultured in microdrops (approximate volume 0-05 ml) in batches of paraffin oil selected for absence of toxicity to cultured embryos (Boots Pure Drug Co., U.K.), under a humid gas mixture of 5 % CO 2, 5 %O 2, and 90 %N 2 at 37 C. The zonae pellucidae were removed with pronase (Calbiochem, U.K., technique of Mintz, 1967). In some 1 Authors' address: Department of Zoology, South Parks Road, Oxford, 0X1 3PS, U.K. 2 Author's address: Laboratoire d'anatomie et d'embryologie Humaines, Rue aux Laines 97, 1000 Bruxelles, Belgium. 3 Reprint requests to C. F. Graham.

2 38 S. J. KELLY, J. G. MULNARD AND C. F. GRAHAM experiments the cells of the embryo were dissociated by gentle pipetting through a flame polished micropipette. Dissociation was sometimes assisted by placing the embryos in Whitten's medium in which the calcium concentration had been lowered to 0-02 mm. Zona-free eggs were cultured either in siliconized glass dishes (siliconized with Repelcote, BDH, U.K.) or in bacteriological grade plastic dishes (Sterilin Ltd., Richmond, U.K.). Eggs with the zona intact were also cultured in plastic tissue culture dishes and flasks (the latter were 25 cm 2 tissue culture flasks, Falcon Plastics, Oxnard, California). For prolonged observations on the microscope stage it was helpful to culture the embryos in sealed flasks to maintain the correct gas mixture ('embryo in bottle' technique). In almost all cases a channelled microscope stage was warmed by water from a thermocirculator (Churchill Ltd., Greenford, Middlesex, U.K.). Cine films were made of embryos cultured in a different medium under different conditions (Mulnard, 1967). Data obtained with this method is indicated in the figure legends. Observations of division asynchrony The cells were observed at regular intervals (see Table legends). Complete embryos (zona on or off) were drawn by eye to record the three-dimensional arrangement of cells. In some cases the embryos were drawn and also photographed with a still camera. The photographic records were useful if the level of focus was recorded. Embryos tend to roll about when contained inside a zona and to avoid confusion, one cell was frequently marked with an oil droplet. The oil was silicone fluid (MS 550, BDH, U.K.), and the injection procedure and drop size were as described by Wilson, Bolton & Cuttler (1972) with some minor changes (Graham & Deussen, 1978). Oil droplets were not required to follow cells in the cine film records. To observe cell division in dissociated embryos dividing to the 16-cell stage, it was necessary to lower the Ca 2+ concentration to 0-0 mm just before the 16-cell stage. This medium reduces adhesion between the cells and cell outlines remain clear. Data on division asynchrony was rejected if the cells did not divide through two further divisions after the division at which the data was collected (about 5 % of the embryos were rejected). Usually both dissociated and intact embryos developed well up to the blastocyst stage (see cell numbers in Table 2). Oil injected cells either lysed in the first hour after injection (about 10 % of the cells) or divided as well as controls up to the blastocyst stage. At this stage trophectoderm cells which contained an oil droplet tended to lyse if the embryo was inside the zona pellucida.

3 Cell allocation and cell division 39 Labelling of cells and reassembly of the embryo The experiments in section 3 of this paper were conducted on embryos which had been dissociated at the - to 7-cell stage. The division of each of the four cells to the 8-cell stage was observed at 30 min intervals. Either the first or the last cell to divide to the 8-cell stage was selected and the pair of daughter cells were immediately labelled for 2 h in [ 3 H]thymidine (specific activity 26 Ci/mM, Radiochemical Centre, Amersham, U.K., technique described by Kelly & Rossant, 1976). They were rinsed in several changes of a 1:1 mixture of Whitten's medium: heat inactivated foetal calf serum during the next hour (Flow Laboratories, Irvine, Scotland). Subsequently they were cultured for 3-5 h in the serum containing medium and then rinsed in Whitten's medium before reassembly. The unlabelled cells were treated similarly. The pairs of cells from each embryo were arranged in two sets of four (Fig. 1 A), and then one set was placed above the other so that each cell made contact with three other cells and so that the centre of each cell was near the corner of a cube (Fig. 1 B). The embryos were then cultured to the blastocyst stage (32-2 h). They were fixed, embedded, and sectioned at ^m (techniques from Hillman, Sherman & Graham, 1972). Line drawings or photographs were made of each section of each blastocyst and the cell numbers in the ICM and the trophectoderm counted. The sections were processed for autoradiography and the autoradiographs were exposed for 2 weeks (Hillman et al. 1972). The numbers and locations of labelled cells were scored. Previously it has been shown that tritiated thymidine does not prevent cells at a similar stage of mouse development from either dividing or differentiating (Kelly & Rossant, 1976). RESULTS The results are arranged in three sections. First, there is data which shows that division asynchrony is a common feature of preimplantation mouse development. Second, there is data which shows that the first cell to divide to the -cell stage within each embryo tends to produce daughter cells which divide ahead of the daughters of the second cell to divide to the -cell stage. Lastly, there is evidence that the first cell to divide to the 8-cell stage contributes more daughter cells to the ICM than does the last cell to divide to the 8-cell stage. 1. Asynchrony of cell division within the embryo Asynchrony of cell division within the embryo was usually obseived between the 2- to -, - to 8-, and 8- to 16-cell stages (Table 1). In this Table, data from a variety of mouse strains were combined because there was no obvious difference between the strains. Data on division in intact embryos was collected in two ways. With long intervals of observations (5-20 min), out of 28 appeared to divide synchronously; this synchrony was probably an artifact of the long

4 0 S. J. KELLY, J. G. MULNARD AND C. F. GRAHAM Table 1. Asynchrony of cell division within the embryo Observations on embryos from C3H/H, (C57BL6x CBA) F 2, A2G, and 129J/Sv. Asterisk marks the cine film observations on the embryos of Swiss albino mice. These embryos were observed at 0 sec intervals. The times of division do not exactly correspond to the intervals of observation. This is because cells were followed continuously under the microscope when the start of cytokinesis was noticed, a indicates that the AB cells (the first to divide) had a significantly shorter 2-cell-stage cell cycle than the CD cells (the second to divide) at the 0-1 % level; b indicates that it was shorter at the 1 % level (related t test, Meddis, 1975). Division 2-A Stage at which culture initiated Intact 2-cell Intact 2-cell Intact 2-cell Zona free 2-cell Zona free 2-cell Zona free 2-cell Intact 2-cell Intact 2-cell Zona free 2-cell Zona free 2-cell Zona free 2-cell Intact -cell Zona free -cell Zona free 2-cell Treatment None None Oil in one cell None Oil in one cell Dissociated None Oil in one cell None Oil in one cell Dissociated None Dissociated Dissociated twice Number asynchronous/ total number 12/1 12/12 12/1 5/5 10/12 1/16 3/3 9/9 6/6 / 15/15 13/13 8/8 7/7 Observation interval (min) Interval from first to last division within asynchronous embryos (min) -A ^ Range i Mean 5 a 68 a a 52 9 b 56 b observation intervals. For when data was collected from cine film (every 0 sec), 12 out of 12 embryos divided asynchronously; one embryo in this series had a division interval of 1 min (the time between completion of division in the first and last cell). For the - to 8- and 8- to 16-cell divisions, all the embryos displayed asynchronous divisions. The mean and range of the time intervals between first and last divisions are given in Table 1. The results show that the first cell to divide to the -cell stage within each embryo usually had a shorter 2-cell-stage cell cycle than its slower partner (statistically significant division asynchrony, see legend to Table 1). The data also showed that neither the various treatments nor the time at which culture was initiated had a marked or consistent effect on the asynchrony which was

5 Cell allocation and cell division 1 observed at the 2- to - and - to 8-cell divisions. It is therefore likely that the asynchrony which was observed at the 8- to 16-cell division (in embryos dissociated at the 2-cell stage and again after the next division) resembled that which occurred in intact embryos developing in culture. Clearly asynchrony of cell division is a regular feature of preimplantation mouse development. 2. Division order and cell cycle durations Division order was observed within the embryos to find out whether the first cell to divide from the 2- to -cell stage (nominated the AB cell) produced daughter cells that divided ahead of the daughters of its slower partner (designated the CD cell, see Gulyas, 1975). The data from various strains were combined because no interstrain differences were observed. 2- to 8-cell divisions The division order to the 8-cell stage is shown in Table 2A. Notice that an AB daughter was usually the first cell to divide to the 8-cell stage (0/8 cases), and in no case did CD produce the two first cells to divide to the 8-cell stage. This pattern of cell division was not disturbed by removal of the zona pellucida, by injection of oil droplets, or by dissociation of the embryo at the 2-cell stage. This last observation suggests that this pattern of division is a property of the individual cells rather than the result of interactions between all the cells of the embryo. The duration of cell cycles between the -cell and 8-cell stages was studied next in an attempt to account for the observed division order. The cell cycle durations of the daughters of AB and of CD are shown in Table 3. This Table contains data from a variety of strains which were exposed to diverse treatments. Nevertheless, the means and trends were similar for all treatments and the data were combined for analysis by the related t test (Meddis, 1975). This test compares the differences in cell cycle duration within each embryo. Several trends emerge from these comparisons. The faster daughter of AB had a significantly shorter -cell stage cell cycle than the slower daughter of AB (significantly different at the 0-1 % level). However, the cell cycle of the faster daughter of AB was just longer than that of the faster CD daughters (significantly different at the 1 %). The cell cycle durations of the faster and slower daughters of CD were not significantly different from each other. Notice that the mean cell cycle of the AB daughters was longer than the mean cell cycle of the CD daughters (significantly different at the 1 % level). The implication is that AB daughters tend to be first to the 8-cell stage simply because they were derived from the cell which was ahead at the 2- to -cell division, and not as a result of an intrinsically shorter cell cycle after the 2-cell stage.

6 2 S. J. KELLY, J. G. MULNARD AND C. F. GRAHAM Table 2. Tendency of AB daughters to divide ahead of CD daughters The embryos used in these experiments were selected for asynchrony at the 2- to - cell division using intervals of observation ranging from 5 to 30 min. The cell divisions in section A (to the 8-cell stage) could be observed in normal culture medium. To observe the cell divisions from the 8- to the 16-cell stage (section B), it was necessary to lower the calcium content of the medium to 00mM and no compensation was made for the slight change in osmolarity. To observe the cell numbers in section C, the embryos were flattened out and stained according to the procedure of Tarkowski (1966). The asterisk draws attention to the cell numbers in the blastocysts derived from AB and CD. These numbers were significantly different at the 25 % level by the related t test (Meddis, 1975). Frequently two cells had divided to a particular cell stage during an interval in observation. Data from these observations are excluded and this exclusion accounts for the variation in the number of embryos along particular lines of Section A of this Table. The embryos in these experiments were from the same strains as those listed in the legend to Table 1. Treatment Number of embryos Mean (range) interval from 2- to -cell stage One AB daughter first to the 8-cell stage Both AB daughters to 8-cell stage before CorD One AB daughter last to the 8-cell stage Zona on Zona on, oil in one cell Zona off Zona off, oil in one cell Zona off, dissociated Totals A. Development to the 8-cell stage (1-180) 11/15-7 (5-135) 3/ (16-120) 5/ (18-125) 6/ (30-157) 15/ /8 5/10 1/ 3/7 3/10 12/23 2/5 6/17 2/ 2/7 2/9 6/26 18/63 B. Development to the 16-cell stage (sequential observations> on nine embryos) Treatment Zona off, dissociated Mean (range) interval from?. tn -rell stage 51-3 (15-105) Frequency with which an AB daughter divided to form an embryo with one of the following cell numbers 9 7/ /9 8/9 A /9 /9 1 2/ /9 2/9

7 Cell allocation and cell division 3 Treatment Morula - Zona off, dissociated Blastocyst - Zona off, dissociated Table 2 (cont.) C. Development to morula and blastocyst Mean (range) interval from 1. tr\ -reil stage 57-3 (30-75) 8- (30-210) Frequency with which ABhad formed more Hoi lovifpr UClUgl I IW.L cells than CD 5/6 13/16 Mean (range) cell number AB daughters 1-5 (12-17) 33-7 (22-5) A CD daughters 116 (9-15) * 31-2 (21-2) - to 16-cell divisions The division order of AB and CD descendants was investigated during the divisions to the 16-cell stage (Table 2B). These observations were made on embryos dissociated at the two-cell stage and again after the next division. This procedure reduced the chances of muddling the cells. The AB descendants were usually the first to divide to the 16-cell stage. In these nine embryos, of the first 36 cells to divide (i.e. the first four in each embryo to bring them all to the 12-cell stage, see Table 2B), 27 were AB descendants. In contrast AB descendants only contribute nine cells amongst the latter 36 cells to divide through to the 16-cell stage. In order to compare statistically this apparent tendency for the AB descendants to reach the 16-cell stage before the CD descendants, a score of eight was allotted to the first cell to divide in each embryo. The second was allotted a score of seven and so on down to a score of one for the last cell to divide. Using these scores, the Wilcoxon matched pairs test (Meddis, 1975) showed that the AB descendants had a significantly higher score than the CD descendants (significantly at the 5 % level T = 5). This shows that the AB descendants have a statistically significant tendency to reach the 16-cell stage before the CD descendants. The duration of the cell cycles was now studied in the hope that these division orders could be explained. Accurate data were only available for seven of these embryos and these are in Table 3 B. This data shows that the mean time taken for the completion of the - and 8-cell stages was not significantly different for the AB and CD descendants (175- and 11-9) or for the fastest AB and the fastest CD descendants (135-0 and ). Again, the implication is that the AB descendants tend to reach the 16-cell stage ahead of the CD descendants simply because they were derived from a cell which was ahead at the 2- to -cell division, and not as a result of an intrinsically shorter cell cycle of their own. However, the durations of this period were variable amongst the AB descendants; within each embryo the fastest AB descendant had a

8 S. J. KELLY, J. G. MULNARD AND C. F. GRAHAM Table 3. Durations of cell cycles A. Duration of the -cell stage Treatment Zona on Zona on, oil in one cell Zona off Zona off, oil in one cell Zona off, dissociated Means Number of embryos (n = 26) Mean (range) interval from 11. \ji to -cell stage (min) 52- (18-110) 2-3 (5-185) 69-7 (16-111) 78-8 (3-168) 7- (15-105) 58-3 f Mean (range) duration of -cell stagt5 in min Faster ( ) 77-7 ( ) 7920 ( ) (78-80) 78-6 ( ) AB daughters Slower 86- ( ) ( ) ( ) ( ) ( ) 82-1 A CD daughters Faster A 71-6 (69-825) 7710 ( ) (700-86) 785 ( ) ( ) Slower (69-910) ( ) 8120 ( ) ( ) 7861 ( ) B. Duration of the - and 8-cell cycles combined (Observations on seven embryos, dissociated at the 2- and the -cell stages. Mean interval 2- to -cell stage was 37-3 min, range was 1-75 min.) Mean (range) duration in min of the - and 8-cell cycles Daughters of AB (order to division) Daughters of CD (order to division) First Second Third Fourth First Second Third Fourth 1350 ( ) ( ) (1285- (1690) ( ) ( ) ( ) ( ) ( ) A.B mean duration = 175- CD mean duration = : 11-9 These observations were made on the same strains of embryos as those indicated in the legend to Table 1. significantly shorter - to 16-cell period than that of the slowest (significant at the 1 % level). This variability was also apparent amongst the descendants of CD; the fastest cell had a significantly shorter period than the slowest cell (significant at the 0-1 % level). Heterogeneity in cell cycle lengths is apparently a feature of individual cells, but does not appear to be inherited as a cell autonomous feature of a particular cell line within the dissociated mouse embryo; this is in contrast to the situation found in embryos with precise cell lineages (van der Biggelaar & Boon Neemeijer, 1973).

9 2-cell stage to morula and blastocyst Cell allocation and cell division 5 The division order to the morula and the blastocyst could not be directly observed. Instead embryos were dissociated at the 2-cell stage and the cell numbers were counted in the half size morulae and blastocysts which developed from the AB and CD cells respectively (Table 2C). The morulae were counted in the morning of the fouilh day of development. They had divided to form embryos, which had they been intact, would have had a mean cell number of 26 (range 2-31). In these six embryos, the AB descendants were more numerous than the CD descendants in five cases. The AB half and the CD half had identical cell numbers in the sixth case. The blastocysts were counted early on the morning of the fifth day of development. They had divided so that, had they been intact embryos, they would have had a mean cell number of 65 (range 9-96). In 13 out of 16 embryos, the descendants of AB were more numerous than the descendants of CD. Overall AB had significantly more descendants than CD (significantly different at the 2-5 % level, related t test). Clearly AB descendants tend to divide ahead of the descendants of CD in all divisions from the -cell to the blastocyst stage. All results except the cine film results were obtained with embryos which were observed to be asynchronous at the 2- to -cell division using relatively long intervals of observation. In no experiment were more than 30 % of the embryos discarded because of synchronous 2- to -cell divisions; usually less than 10% were discarded. Our observations therefore relate to the majority of embryos in any batch. 3. Relationship between division order and cell allocation Division order might affect the process of allocation of cells to the ICM and to the trophectoderm of the blastocyst. This was investigated by looking for associations between division order to the 8-cell stage and the contribution of cells to the two tissues of the blastocyst. Embryos were dissociated into single cells at the -cell stage and the division order to the 8-cell stage was observed. Within each embryo the daughters of either the first or the last cell to divide to the 8-cell stage were labelled with tritiated thymidine. Next the embryos were reassembled in such a way that all the eight cells were in similar positions relative to each other (Fig. 1, and Materials and Methods). From our previous observations it was probable that the first cell to divide to the 8-cell stage would be a daughter of AB and that the last cell to do so would be a daughter of CD; our procedure therefore allowed us to follow some of the daughters of AB and CD through to the blastocyst stage. First it is necessary to assess the effect of the labelling procedure on the results. Since two cells were labelled at the 8-cell stage, these should divide to form 25 % of the cells in the blastocyst. Tables and 5 show that the mean percentage of labelled cells was less than expected - 21 % (significantly different atp = EMB 8

10 6 S. J. KELLY, J. G. MULNARD AND C. F. GRAHAM Fig. 1. Re-assembly of labelled embryos. All the cells from a dissociated -cell embryo were kept apart and divided to the 8-cell stage. The pairs of cells were arranged in two sets of four (Figure 1 A). When these sets had adhered, one set was placed on top of the other (Figure 1B). Scale bar = 50 /*m. Table. Labelled and unlabelled cell counts of blastocysts in which the progeny of the first cell to cleave away from the -cell stage were labelled for 2 h in [ 3 H]thymidine Embryo no. Total cell no. No. in ICM ICM/total r " Total Labelled cells ICM ICM/total Total no. labelled/ total no. No. labelled in ICM/total no. in ICM Mean Ranges t Data obtained on mice from the PO and C57BL6 strains. Comparisons were made of the differences between the figures appearing in columns 8 and 7 and columns 6 and 3 (see text), and those obtained between the same columns in Table 5. Blastocysts have been arranged in order of total cell number.

11 Cell allocation and cell division 7 Table 5. Labelled and unlabelled cell counts of' blastocysts in which the progeny of the last cell to cleave away from the -cell stage were labelled for 2 h in [ 3 H]thymidine Embryo no. Total cell no. No. in 1CM ICM/total Labelled cells A Total ICM ICM/total Total no. labelled/ total no. No. labelled in ICM/total no. in ICM Means Ranges Data obtained on mice from the PO and C57BL6 strains. Comparisons were made as described in the legend to Table. The blastocysts have been arranged in order of total cell number. for 23 D.F., Student's t test, Bailey, 1959). Tritiated thymidine appears to slow the rate of cell division and might therefore obscure the phenomenon under investigation. The degree of retardation, however, was similar when either the first or the last pair of cells to the 8-cell stage were labelled (20-5 % does not differ significantly from 21-5%). It was subsequently assumed that any effect of the tritiated thymidine on cell allocation would be the same in both series of experiments and that it was legitimate to look for differences between the series. The distribution of labelled cells in blastocysts formed from embryos in which the daughters of the first cell to divide to the 8-cell stage were labelled is given in Table, and that for the blastocysts formed from embryos in which the daughters of the last cell to divide to the 8-cell stage were labelled is given in Table 5. These tables also show the calculated proportions used in the analyses of the distribution of the labelled cells. If the labelled cells were distributed randomly in the blastocyst, then the proportion of labelled cells amongst the total number of cells in the ICM (column 8) should be the same as the proportion of the labelled cells in the blastocyst as a whole (column 7) (comparison 1) Another way of looking at the distribution of labelled cells is to compare the proportion of the total number of labelled cells which appear in the ICM (column 6) to the proportion of the total cell number that appear in the ICM (column 3) (second comparison); this comparison makes some allowance for -2

12 8 S. J. KELLY, J. G. MULNARD AND C. F. GRAHAM the individual variation in the size of the ICM. Deviations from a random distribution will produce differences between these proportions in each case. If the progeny of the first cell to cleave away from the -cell stage have a significantly increased chance of appealing in the ICM over that of the progeny of the last cell to cleave away from the -cell stage, then the differences between these proportions in the first series should differ significantly from the differences between them in the second series. These differences were compared by performing two sample t tests (Bailey, 1959) on the two comparisons in each series. (These tests were legitimate because the variances of the two samples were similar in each series). The test showed that for both comparisons there was a statistically significantly greater contribution of the progeny of the first cell to divide away from the -cell stage to the ICM than of the progeny of the last cell to divide (significant at P = 01 for the first comparison and at P = 0-05 for the second). Clearly there is an association between division order and the contribution of cells to the ICM. Notice that there is no evidence that the first cell to divide to the 8-cell stage has formed more daughters than the last cell to divide to the 8-cell stage. This is possibly because the blastocysts were fixed at various times without any attempt to match the two series. DISCUSSION AB and CD cell division Chance processes could account for the observation that one cell of the 2-cell embryo divides to the -cell stage before the other; this is probably not the case because we have noticed that there is some regularity in the orientation of the cell which divided first (unpublished observations). Lewis & Wright (1935) believed that the first cell to divide was the larger cell produced by unequal first cleavage. This relationship was not noticed in this work (see also Mulnard, 1967) although the first cell to divide appeared to increase in size just before division. Early in the 2-cell stage, the two cells do in fact differ slightly in dry mass and this difference increases a little during interphase (Abramczuk & Sawicki, 197). There are other signs that the two cells are metabolically distinct: the nucleoli may differ in the time at which they acquire staining properties which are thought to indicate the presence of ribosomal RNA synthesis (Engel, Zenzes & Schmid, 1977). and they may also differ in the duration of DNA synthesis (Luthardt & Donahue, 1975). Possibly the metabolism of the two cells becomes different during the 2-cell stage. It appears that the AB cells do not transmit to their daughters the character of short cell cycles when they are grown in isolation from the CD cells. Thus AB daughters do not have a shorter cell cycle than CD daughters at the -cell stage. Similarly the period from the - to the 16-cell stage is not shorter for AB daughters; in fact it is slightly longer. The observed regularity of division order

13 Cell allocation and cell division 9 during development then appears to be the consequence of two processes: there is the initial asynchrony at the 2- to -cell division followed by similar mean cell cycle times for AB and CD descendants, which do not obscure this asynchrony. There is therefore no evidence for a transmissible state which segregates at the 2-cell stage of development. We do not have sufficient evidence to decide if such a state segregates at the -cell stage; cell cycle heterogeneity is observed at all stages. Our observations on the division order to the 8-cell stages confirm previous suggestions by Lewis & Wright (1935) and Borghese & Cassini (1963). A (locations of cells to the ICM and to the trophectoderm Previously it had been noticed that the first cells to divide to the 16-cell stage tended to form inside cells in intact cultured embryos (Barlow, Owen & Graham, 1972). It follows from our data on division order that these cells were usually the products of AB. The labelling experiments in this paper show that within an embryo, the first cell to divide to the 8-cell stage tends to contribute more cells to the ICM than does the last cell to divide the 8-cell stage. Since the first cell to divide to the 8-cell stage is usually derived from AB (in 0/8 cases), it is likely that one -cell-stage daughter of AB contributes disproportionately more cells to the ICM than does one -cell-stage daughter of CD. Our experimental procedure may obscure an even more regular pattern in the intact embryo. First, our observations were made on embryos which were dissociated at the -cell stage and reassembled at the 8-cell stage. Any relationship between division order and contribution to the ICM which depends on mechanisms which operate before reassembly of the embryo was therefore excluded. There is for instance a relationship between division order and cell position in the intact embryo at the 8-cell stage (Graham & Duessen, 1978). Second, we did not follow the daughters of AB and CD separately through from the 2-cell stage in these labelling experiments, and some of the daughters of CD may have been amongst the first cells to the 8-cell stage. Third, the labelling procedure slowed the division of the labelled cells. Fourth, the cell arrangements in the reassembled embryos were different from those in intact embryos where the daughters of each cell at the -cell stage are in different relative positions; in the reassembled embryos the cells were in identical positions relative to each other. Despite all these difficulties, our results demonstrate that there is a relationship between division order to the 8-cell stage and the contribution of of cells to the ICM. This relationship must depend in these experiments solely on mechanisms which operate after the reassembly of the embryos. In the reassembled embryos, the first and the last cell to divide to the 8-cell stage together formed about an half of the cells in the ICM. It is likely that the second and the third cell to divide to this stage also contributed to the ICM. Our observations confirm the studies of Wilson et al. (1972) which showed that no cell at the 2- and -cell stage contributed exclusively to the ICM in intact

14 50 S. J. KELLY, J. G. MULNARD AND C. F. GRAHAM embryos. Each cell of a -cell embryo has previously been shown to be able to form all the tissues of an adult mouse (Kelly, 1977), and our results suggest that each cell of a -cell embryo contributes to the 1CM in these reassembled embryos. CONCLUSIONS 1. One of the cells in the 2-cell embryo is the first to divide to the -cell stage (AB cell) and its daughters tend to divide ahead of those cells derived from its slower partner at all subsequent stages of development up to the blastocyst stage. 2. AB descendants do not have shorter cell cycles than the other cells of the embryo during subsequent stages of development. 3. The first cell to divide to the 8-cell stage tends to contribute disproportionately more descendants to the 1CM when compared to the last cell to divide to the 8-cell stage. We would like to thank A. J. Copp, J. Haywood, R. L. Gardner, V. E. Papaioannou and J. West for helpful discussions. The MRC kindly supported these studies. REFERENCES ABRAMCZUK, J. & SAWICKI, W. (197). Variation in dry mass and volume of nonfertilized oocytes and blastomeres of 1-, 2-, and -celled mouse embryos. /. exp. Zool. 188, BAILEY, N. J. J. (1959). Statistical Methods in Biology. London: English Universities Press. BARLOW, P., OWEN, D. A. S. & GRAHAM, C. F. (1972). DNA synthesis in the preimplantation mouse embryo. /. Embryol. exp. Morph. 27, BORGHESE, E. & CASSINI, A. (1963). Cleavage of mouse egg. In Cinemicrography in Cell Biology (ed. G. G. Rose), pp New York and London: Academic Press. DALCQ, A. M. (1957). Introduction to General Embryology. Oxford University Press. ENGEL, W., ZENZES, M. T. & SCHMID, M. (1977). Activation of mouse ribosomal RNA genes at the 2 cell stage. Human Genet. 38, GRAHAM, C. F. & DEUSSEN, Z. A. (1978). Features of cell lineage in preimplantation mouse development. /. Embryol. exp. Morph. (In the Press.) GULYAS, B. J. (1975). A re-examination of cleavage patterns in eutherian mammalian eggs: rotation of blastomere pairs during second cleavage in the rabbit. J. exp. Zool. 193, HILLMAN, N., SHERMAN, M. I. & GRAHAM, C. F. (1972). The effect of spatial arrangement on cell determination during mouse development. /. Embryol. exp. Morph. 28, KELLY, S. J. (1977). Studies on the developmental potential of - and 8-cell stage mouse blastomeres. /. exp. Zool. 200, KELLY, S. J. & ROSSANT, J. (1976). The effect of short term labelling in [ 3 H]thymidine on the viability of mouse blastomeres: alone and in combination with unlabelled blastomeres. /. Embryol. exp. Morph. 35, LEWIS, W. H. & WRIGHT, W. S. (1935). On the early development of the mouse egg. Contr. Embryol. Carnegie Instn., Washington 18, LUTHARDT, F. W. & DONAHUE, R. P. (1975). DNA synthesis in developing 2-cell mouse embryos. Devi Biol., MEDDIS, R. (1975). Statistical Handbook for Non-statisticians. McGraw-Hill. MINTZ, B. (1967). Mammalian embryo culture. In Methods in Developmental Biology (ed. F. H. Wilt & N. K. Wessels), pp New York: T. Y. Corwell. MULNARD, J. G. (1967). Analyse microcinematographique du development de l'oeuf de souris du stade II au blastocyste. Archs Biol. (Liege) 78,

15 Cell allocation and cell division 51 TARKOWSKT, A. K. (1966). An air drying method for chromosome preparations from mouse eggs. Cytogenetics 5, VAN DEN BIGGELAAR, J. A. M. & BOON-NEEMIEJER, E. K. (1973). Origin and prospective significance of division asynchrony during early molluscan development. In The Cell Cycle in Development and Differentiation (ed. M. Balls & F. S. Billett), pp Cambridge University Press. WHITTEN, W. K. (1971). Nutrient requirements for the culture of preimplantation embryos in vitro. Advances in the Biosciences 6, (ed. G. Raspe), Oxford: Pergamon Press. WILSON, I. B., BOLTON, E. & CUTTLER, R. H. (1972). Preimplantation differentiation in the mouse egg as revealed by microinjection of vital markers./. Embryol. exp. Morph. 27, (Received 16 February 1978)

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