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inserted in the plantar region of the paw and a Laser doppler ¯owmeter probe applied tothe plantar surface (Permided PF2B) was used to measure basal and capsaicin-evokedchanges. Student's t-test was used and differences were considered signi®cant at P , 0.05.

Behavioural studies

Between 6 and 13 adult male F2-3 129SV´Balb/c mice of each genotype were used for allstudies. Thermal11, mechanical11, chemical11, visceral11 sensitivity, stress-inducedanalgesia27 and neuropathic pain29,30 was assessed as described previously. Tail-¯ick latency(by directing a concentrated light beam to the tail of the mouse) was monitored before andafter intrathecal injection of 10 mg NPY. For Evans blue plasma extravasation the followingwere used: capsaicin, 3 mg in 10 ml (Sigma; dissolved in 5% ethanol, 5% Tween-80 and90% saline); 1% carrageenan (Sigma; dissolved in saline); SP, 50 pmol per paw (Sigma;dissolved in saline); 5% mustard oil (Fluka; dissolved in mineral oil); NPY Y1 receptoragonist [Leu31-Pro34]-NPY, 10 mg per paw (Calbiochem; dissolved in saline and 5% aceticacid); and NPY Y1 receptor selective antagonist BIBP 3226, 10 mg kg-1 in 10 ml kg-1

(American Peptide; dissolved in saline and 5% acetic acid). Brie¯y, mice were anaesthe-tized and injected intravenously with Evans blue (50 mg kg-1) into the jugular vein. Theabove agents were injected into one paw of the animal except for the Y1 receptor selectiveanatagonist BIBP 3226, which was injected intravenously 10 min before injection into thepaw. The other paw was injected with vehicle. After 30 min the plantar skin of the paw wasremoved, dried of excess liquid, weighed and incubated in formamide for 24 h at 56 8C.Extravasated Evans blue was measured by spectrophotometer at 620 nm. Mechanicalsensitivity was determined before, 30 min and 3 h after capsaicin and carrageenanadministration, respectively. Carrageenan in¯ammation was induced similarly butextravasation was measured after 4 h. The paw diameter was measured before and aftercapsaicin, carrageenan or vehicle administration using a spring-loaded calliper.

Immunohistochemistry

Wild-type and Y1-/- mice were perfused with 4% paraformaldehyde (for SP receptorimmunohistochemistry, mice were perfused with 4% paraformaldehyde and 12.5% picricacid 10 min after capsaicin injection into hindpaw), and the spinal cord and dorsal rootganglia were sectioned coronally (15 mm in thickness). Capsaicin was injected intrader-mally into dorsal skin of mice. After 10 min the skin was removed, post®xed and sectionedas above. Immunohistochemistry was done as described28 using anti-b-galactosidase(1:200 dilution, ICN/Cappel), rabbit anti-SP (1:5,000 dilution, Chemicon), guinea-piganti-SP (1:200 dilution, Peninsula Lab.), rhodamine-conjugated bandeiraea simplicifolialectin I (Isolectin B4; 1:100 dilution, Vector), anti-NPY (1:200 dilution, Peninsula Lab.),and rhodamine or FITC-conjugated secondary antisera (Jackson). For SP receptorimmunohistochemistry, sections were incubated for 30 min in PBS, 50% methanol, and0.6% H2O2 before incubation in 10% goat serum. The antiserum (Chemicon 1:2,000) wasused in the ¯uorescein TSA ¯uorescence system (NEN). b-galactosidase histochemicalstaining was done as described28.

EIA

Capsaicin or saline was injected into the paw of wild-type or Y1-/-mice. After 10 min, thepaw was removed and the skin was cut open and washed in PBS and 0.1% BSA for 10 min.The skin was then dried, weighed, transferred to a new container, and frozen. The liquidwas centrifuged at 4,000 r.p.m. for 15 min. Supernatant was transferred to a new tube,weighed and frozen. The lumbar part of spinal cord was removed, weighed and frozen. Thesamples were then assayed for SP according to the manufacturer's instructions using SPhigh sensitivity EIA kit (Peninsula Lab.).

Received 29 September; accepted 13 November 2000.

1. Munglani, R., Hudspith, M. J. & Hunt, S. P. The therapeutic potential of neuropeptide Y. Analgesic,

anxiolytic and antihypertensive. Drugs 52, 371±389 (1996).

2. Hua, X. Y. et al. The antinociceptive effects of spinally administered neuropeptide Y in the rat:

systematic studies on structure-activity relationship. J. Pharmacol. Exp. Ther. 258, 243±248 (1991).

3. Duggan, A. W., Hope, P. J. & Lang, C. W. Microinjection of neuropeptide Y into the super®cial dorsal

horn reduces stimulus-evoked release of immunoreactive substance P in the anaesthetized cat.

Neuroscience 44, 733±740 (1991).

4. Hudspith, M. J. & Munglani, R. Neuropeptide Y: friend or foe. Eur. J. Pain 3, 3±6 (1999).

5. Pedrazzini, T. et al. Cardiovascular response, feeding behavior and locomotor activity in mice lacking

the NPY Y1 receptor. Nature Medicine 4, 722±726 (1998).

6. Naveilhan, P., Neveu, I., Arenas, E. & Ernfors, P. Complementary and overlapping expression of Y1, Y2

and Y5 receptors in the developing and adult mouse nervous system. Neuroscience 87, 289±302

(1998).

7. Zhang, X. et al. Localization of neuropeptide Y Y1 receptors in the rat nervous system with special

reference to somatic receptors on small dorsal root ganglion neurons. Proc. Natl Acad. Sci. USA 91,

11738±11742 (1994).

8. Snider, W. D. & McMahon, S. B. Tackling pain at the source: new ideas about nociceptors. Neuron 20,

629±632 (1998).

9. Allen, B. J. et al. Primary afferent ®bers that contribute to increased substance P receptor

internalization in the spinal cord after injury. J. Neurophysiol. 81, 1379±1390 (1999).

10. Mantyh, P. W. et al. Receptor endocytosis and dendrite reshaping in spinal neurons after somato-

sensory stimulation. Science 268, 1629±1632 (1995).

11. Cao, Y. Q. et al. Primary afferent tachykinins are required to experience moderate to intense pain.

Nature 392, 390±394 (1998).

12. Watkins, L. R. & Mayer, D. J. Multiple endogenous opiate and non-opiate analgesia systems: evidence

of their existence and clinical implications. Ann. N. Y. Acad. Sci. 467, 273±299 (1986).

13. Rubinstein, M. et al. Absence of opioid stress-induced analgesia in mice lacking b-endorphin by site-

directed mutagenesis. Proc. Natl Acad. Sci. USA 93, 3995±4000 (1996).

14. Marek, P., Mogil, J. S., Sternberg, W. F., Panocka, I. & Liebeskind, J. C. N-methyl-D-aspartic acid

(NMDA) receptor antagonist MK-801 blocks non-opioid stress-induced analgesia. II. Comparison

across three swim-stress paradigms in selectively bred mice. Brain Res. 578, 197±203 (1992).

15. Zhang, X., Wiesenfeld-Hallin, Z. & Hokfelt, T. Effect of peripheral axotomy on expression of

neuropeptide Y receptor mRNA in rat lumbar dorsal root ganglia. Eur. J. Neurosci. 6, 43±57 (1994).

16. Xu, X. J., Hao, J. X., Hokfelt, T. & Wiesenfeld-Hallin, Z. The effects of intrathecal neuropeptide Y on

the spinal nociceptive ¯exor re¯ex in rats with intact sciatic nerves and after peripheral axotomy.

Neuroscience 63, 817±826 (1994).

17. Jancso, N., Jancso-Gabor, A. & Szolcsanyi, J. Direct evidence for neurogenic in¯ammation and its

prevention by denervation and by pretreatment with capsaicin. Br. J. Pharmacol. 31, 138±151 (1967).

18. Jancso, N., Jancso-Gabor, A. & Szolcsanyi, J. The role of sensory nerve endings in neurogenic

in¯ammation induced in human skin and in the eye and paw of the rat. Br. J. Pharmacol. 33, 32±41

(1968).

19. Inoue, H., Asaka, T., Nagata, N. & Koshihara, Y. Mechanism of mustard oil-induced skin

in¯ammation in mice. Eur. J. Pharamacol. 333, 231±240 (1997).

20. Semos, M. L. & Headley, P. M. The role of nitric oxide in spinal nociceptive re¯exes in rats with

neurogenic and non-neurogenic peripheral in¯ammation. Neuropharmacology 33, 1487±1497

(1994).

21. Lembeck, F. & Holzer, P. Substance P as neurogenic mediator of antidromic vasodilation and

neurogenic plasma extravasation. Naunyn Schmiedelbergs Arch. Pharmacol. 310, 175±183 (1979).

22. Shi, T. J., Zhang, X., Berg, O. G., Erickson, J. C., Palmiter, R. D. & Hok¯t, T. Effect of peripheral

axotomy on dorsal root ganglion neuron phenotype and autotomy behaviour in neuropeptide

Y-de®cient mice. Regul. Pept. 25, 161±173 (1998).

23. Wilcox, G. L. Excitatory Neurotransmitters and Pain (eds Bond, M. R., Charlton, J. R. & Woolf, C. J.)

97±117 (Elsevier, New York, 1991).

24. Walker, M. W., Ewald, D. A., Perney, T. M. & Miller, R. J. Neuropeptide Y modulates neurotransmitter

release and Ca2+ currents in rat sensory neurons. J. Neurosci. 8, 2438±2446 (1988).

25. Polgar, E., Shehab, S. A., Watt, C. & Todd, A. J. GABAergic neurons that contain neuropeptide Y

selectively target cells with the neurokinin 1 receptor in laminae III and IV of the rat spinal cord.

J. Neurosci. 19, 2637±2646 (1999).

26. Parker, D. et al. Co-localized neuropeptide Y and GABA have complementary presynaptic effects on

sensory synaptic transmission. Eur. J. Neurosci. 10, 2856±2870 (1998).

27. De Felipe, C. et al. Altered nociception, analgesia and aggression in mice lacking the receptor for

substance P. Nature 392, 394±397 (1998).

28. Naveilhan, P. et al. Normal feeding behavior, body weight and leptin response require the

neuropeptide Y Y2 receptor. Nature Medicine 5, 1188±1193 (1999).

29. Seltzer, Z., Dubner, R. & Shir, Y. A novel behavioral model of neuropathic pain disorders produced in

rats by partial sciatic nerve injury. Pain 43, 205±218 (1990).

30. Malmberg, A. B. & Basbaum, A. I. Partial sciatic nerve injury in the mouse as a model of neuropathic

pain: behavioral and neuroanatomical correlates. Pain 76, 215±222 (1998).

Acknowledgements

We thank L. Klevenvall-Fridvall for technical assistance and L. Johansson for secretarialassistance. This research was supported by the Swedish Medical Research Council theBiotechnology Program of the European Union and the Swedish Cancer Society.

Correspondence and requests for material should be addressed to P.E.(e-mail: patrik@cajal.mbb.ki.se).

.................................................................Role for sperm in spatial patterningof the early mouse embryoKarolina Piotrowska & Magdalena Zernicka-Goetz

Wellcome/CRC Institute and Department of Genetics, University of Cambridge,

Tennis Court Road, Cambridge CB2 1QR, UK

..............................................................................................................................................

Despite an apparent lack of determinants that specify cell fate,spatial patterning of the mouse embryo is evident early indevelopment. The axis of the post-implantation egg cylinder canbe traced back to organization of the pre-implantation blastocyst1.This in turn re¯ects the organization of the cleavage-stage embryoand the animal±vegetal axis of the zygote2,3. These ®ndingssuggest that the cleavage pattern of normal development maybe involved in specifying the future embryonic axis; however,how and when this pattern becomes established is unclear. Inmany animal eggs, the sperm entry position provides a cue forembryonic patterning4±6, but until now no such role has beenfound in mammals. Here we show that the sperm entry positionpredicts the plane of initial cleavage of the mouse egg and cande®ne embryonic and abembryonic halves of the future blasto-

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cyst. In addition, the cell inheriting the sperm entry positionacquires a division advantage and tends to cleave ahead of itssister. As cell identity re¯ects the timing of the early cleavages,these events together shape the blastocyst whose organization willbecome translated into axial patterning after implantation. Wepresent a model for axial development that accommodates these®ndings with the regulative nature of mouse embryos.

Early mouse development is highly regulative and consequentlyaxis formation has been treated as being independent of any aspectof organization of the egg. Thus, there has been no incentive tostudy correlations between the sperm entry position (SEP) anddevelopmental patterns. As this viewpoint has now been ques-tioned1±3, we wanted to determine whether there are any develop-mental consequences of sperm entry on the subsequent patterningof the mouse embryo.

We therefore developed an enduring marker for the SEP. Atransitory protrusion, the fertilization cone, appears at the SEPabove the condensed chromatin of the sperm, but is not detectableafter the sperm head has decondensed and formed the pronucleus(Fig. 1a, b). We found that the SEP could be marked by phyto-haemagglutinin-coated ¯uorescent beads introduced under thezona pellucida and placed in contact with the plasma membrane atthe fertilization cone. Beads remained adhered to the plasma mem-brane throughout subsequent development without undergoingendocytosis.

To determine whether any ¯uidity of the plasma membranemight preclude the use of such beads as positional markers, we®rst ascertained whether they would maintain their relativepositions with respect to the second polar body (PB), a marker ofthe animal pole of the egg until the blastocyst stage2,3 (Fig. 1). Wefound that when beads were positioned near to the PB theyremained in this position in 82% (47/57) of embryos throughoutthe two-cell, morula and blastocyst stages. Similarly, when placedopposite the PB, beads retained their position relative to the PBthroughout these stages in 85% (41/48) of zygotes. In the twogroups, a small proportion of zygotes (18% and 15% respectively)showed relative movement of the beads away from their originalposition. This might be explained by the previously observedrotation of blastomeres after cleavage divisions7 and was seen as aslight displacement of the bead at the two-cell stage. After suchdisplacement, however, the new position of the bead was sub-sequently maintained with respect to the PB until the blastocyststage. Therefore, when the surface of the egg is marked in this way,its features (the PB and the bead) retain their relative positionsthroughout pre-implantation development in most embryos, andthus can be used as landmarks for the animal pole of the zygote andthe site of sperm entry, respectively.

We used beads to mark the SEPs of newly fertilized zygotes tofollow their positions in the subsequent development of theembryo. Differential development is ®rst seen in the two-cell

embryo in the form of asynchrony of the second cleavage divisions.To determine whether there was any relationship between the orderof cleavage and the SEP, we examined the position of the bead inthree-cell embryos in which one of the second cleavages had justoccurred. We found that in 75% of a group of 92 three-cell embryosthe bead marked one of the two smaller cells, indicating that theblastomere inheriting the SEP had divided ahead of its sister(Fig. 1c). This suggests that the incoming sperm can provideinformation to its host egg cytoplasm that in¯uences the order inwhich cells divide.

We then allowed these embryos to develop into blastocysts todetermine whether the SEP would come to lie in any speci®c region,scoring its position ®rst with respect to the animal±vegetal (A±V)axis. The sperm can enter the egg at any point on its surface with theexception of an area above the metaphase II spindle. This is re¯ectedin the distribution of the SEP marker along the A±V axis in a groupof 73 zygotes in which sperm entered in the animal third in 16% ofcases, the equatorial third in 37%, and the vegetal third in 47%.From 50 blastocysts that developed from these zygotes with anintact PB on the A±V axis, 16% had the bead in the animal third,32% in the equatorial region, and 52% in the vegetal third. Thiscorresponds to the proportion of zygotes that had their SEPspositioned in these regions, con®rming that landmarks on the

a b c dPB PB PB PB

ABEm

A

V

fp

mp

FC

Figure 1 Landmarks in the development of the pre-implantation embryo. a, Egg shortly

after fertilization, showing the fertilization cone and the PB. b, Egg after migration of the

male pronucleus (mp) and female pronucleus (fp) towards the centre of the zygote.

c, Three-cell-stage embryo in which one of the second cleavage divisions has taken

place. One of the two small cells is marked with a bead that was placed on the zygote at

the SEP. d, Blastocyst shown in a comparable orientation to the eggs in a±c to indicate its

two principal axes, A±V and Em±Ab. The A±V axis of the blastocyst lies along the medial

region of the blastocyst tangential to the blastocyst cavity (blastocoel) and re¯ects the A±V

axis of the zygote2,3. The blastocoel itself occupies the abembryonic hemisphere. Scale

bar in all ®gures, 20 mm.

10/57 (17%)

Initial position of thebead in the zygote

Spermentryposition

a

b

c

Vegetalpole(control)

Equatorialregion(control)

22/48 (46%)

11/23 (48%)

42/57 (74%)

Position of the bead on Em–Ab axis of blastocyst

22/48 (46%)

8/23 (35%)

5/57 (9%)

4/48 (8%)

4/23 (17%)

Figure 2 The SEP predicts the Em±Ab axis of the blastocyst. a, Zygotes were marked with

beads at the SEP and examined at the blastocyst stage. The position of the bead was

scored depending on whether it lay in the embryonic (left), medial (middle), or

abembryonic region (right). Representative micrographs of such blastocysts are shown in

which the limits of the medial regions are indicated by dashed white lines. b, c, Controls in

which the beads were placed either at the vegetal pole (b) or in the equatorial region (c),

and scored as above.

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zygote retain their relative positions in the blastocyst.We next analysed the distribution of the SEP along the embryonic±

abembryonic (Em±Ab) axis of the blastocyst. This lies orthogonalto the A±V axis, and is morphologically asymmetric in that theblastocyst cavity occupies the abembryonic hemisphere (Fig. 1d).Analysis of the position of the beads showed that their distributionalong this axis was remarkably non-random. To map the positionof the bead on the blastocyst, we de®ned the medial region of theEm±Ab axis to be within three bead diameters (roughly 12.5% ofthe embryo diameter) below or above the blastocoel roof (Fig. 2)and found that in 74% of cases the bead came to lie in this region.This shows that in most embryos the SEP comes to lie near theborder of the embryonic and abembryonic hemispheres.

To investigate the possibility that the bead tends to becomelocated in this region as a result of some physical constraint bearingno relationship to the SEP, we analysed two sets of control experi-ments in which beads were placed on the surface of the zygote atpositions that may or may not have included the SEP. The ®rst setwas from the experiment described above in which beads wereplaced opposite the PB of the zygote and followed to the blastocyst.We found that for the 48 embryos analysed, the bead lay in theembryonic region in 46% of cases, in the medial region in 46%, andin the abembryonic region in 8% (Fig. 2, control 2). We also usedbeads to mark the zygote surface at a random position within theequatorial region. In these experiments beads came to lie in theembryonic region in 48% of blastocysts, in the medial region in35%, and in the abembryonic region in 17% (Fig. 2, control 3). Thuswhen beads are placed at sites without respect to the SEP, they showsigni®cantly different distributions (x2 test, P , 0.001, 2 degrees offreedom) with a reduced tendency to lie in the medial region of theblastocyst. Hence by marking the Em±Ab boundary, the SEPpredicts the orientation of the Em±Ab axis with which it alignsorthogonally.

We considered whether this tendency of the SEP to lie in aparticular region of the blastocyst might re¯ect some earlier devel-opmental event such as the pattern of early cleavage. It is known thatthe ®rst cleavage plane of the zygote is meridional, that is, parallel tothe A±V axis of the zygote as de®ned by the PB at its animal pole.However, the orientation of this initial cleavage about the A±V axishas been thought to be random and not speci®ed by any earlierdevelopmental event3. To determine whether the SEP bears arelationship to this initial cleavage we followed the developmentof 178 zygotes. First we marked the SEP and estimated the positionof the bead with respect to the A±Vaxis before the fertilization conedisappeared and con®rmed its position when the male pronucleushad formed but not yet migrated to the egg centre. In the zygote werecorded the SEP in one of three sectors: animal, equatorial andvegetal (see Fig. 3).

Next we examined the position of the bead shortly after comple-tion of the ®rst division, and subsequently at a more advanced two-cell stage. At the two-cell stage, we further de®ned its positionwithin one of three equal sectors: central, middle and lateral. As the®rst cleavage is meridional with respect to the PB, these sectors lieorthogonal to those in the zygote. If the SEP and the plane of the ®rstcleavage were random with respect to each other, we would expectthe bead to lie with equal probability (33%) in each of these threeregions. However, we found that just after the ®rst cleavage the SEPtended to be at or in close proximity to the furrow separating thecells. In 178 two-cell embryos analysed, the SEP was localized to thecentral zone in 60% of embryos, and in the middle and lateralregions in only 19% and 21%. When those embryos (107 of 178)that showed a central location of SEP were further analysed, roughlyequal numbers had SEP originating from the animal (60%),equatorial (57%) and vegetal (67%) regions of the zygote. Beads

a

b

e

c

f

d

g

Middle

Equatorial

Animal

VegetalCentral Lateral

Figure 3 The SEP predicts the ®rst cleavage plane. a, Sectors in which the position of the

bead was scored in the zygote and the two-cell stage. b±d, Micrographs showing the ®rst

cleavage of an egg marked at its SEP. In this example, the bead is near the vegetal pole of

the zygote (b), is at the furrow during cleavage (c), and remains on the furrow that

separates the cells (d). e±g, A second example of an egg in which the bead is located at

the position of the forming furrow during cleavage (f), but was displaced away from the

furrow that later separates the two cells (g).

Central–40

–20

0

12.5%

–12.5%

20

40

Middle Lateral

a

b

c

Figure 4 Relationship between the SEP at the two-cell stage and its fate at the blastocyst

stage. Zygotes marked at their SEP were classi®ed into three categories after their ®rst

cleavage, depending on whether the bead lay in the central, middle or lateral sectors of

1/2 blastomeres. Shown are representative micrographs of two-cell embryos in which the

bead occupied either the boundary zone (b) or was localized to either the embryonic (a) or

abembryonic pole (c). Zygotes were allowed to develop to the blastocyst and the ®nal

position of the bead was determined. The value plotted here is the distance of the bead

from the border of the embryonic and abembryonic regions delineated by the roof of the

blastocoel projected onto a ¯at plane and assessed as a percentage of the Em±Ab

diameter of each blastocyst. The boundary zone is shown as the area within 12.5% on

each side of the blastocoel roof.

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that were placed randomly in the equatorial regions (not necessarilyexcluding the SEP) of 28 zygotes showed no signi®cant tendency oflocalization to the cleavage furrow: 38% were in the central, 42% inthe middle, and 20% in the lateral regions. These ®ndings show thatirrespective of the position of the SEP in the zygote, there is a strongtendency for the SEP to be localized to the central region near thecleavage plane of the two-cell embryo.

As some rotation of cells can occur after the ®rst cleavage, wedetermined the extent to which the position of the furrow ®nallyseparating the two cells corresponded to the actual site of cyto-kinesis. By catching 37 embryos directly as the ®rst cleavage divisionwas in progress, we found the bead at or very close to the cleavagefurrow in 78% (29/37) of cases (Fig. 3c, f). Unexpectedly, the beadappeared to indicate the plane of cleavage more accurately than didthe PB, as only four of these embryos (11%) had their PBs near tothe cleavage furrow. When these 37 embryos were observed 2±3 hlater, there had been a slight reorientation of blastomeres such thatthe PB was now located in the furrow between the two cells. If thebead and the PB were attached to the same blastomere, this rotationwas able to shift the bead slightly away from the furrow separatingthe cells (Fig. 3f, g). Direct observation of these embryos suggestedthat this rotation could occur within minutes of the completion ofcytokinesis. Thereafter over the next several hours there was verylittle blastomere rotation. Thus, there is a strong correlationbetween the position of sperm entry and the ®rst cleavage plane,and this is still evident even after some blastomeres have undergonerotation.

To determine the relationship between the SEP, the plane of the®rst cleavage and the Em±Ab axis, we cultured a set of embryosfrom the above experiments to the blastocyst stage. To minimizedevelopmental disturbances we examined a subgroup of embryosthat had been observed twice during their early development:initially to con®rm the position of the bead, and then shortlyafter the ®rst cleavage. A total of 50 such blastocysts were scoredin three groups depending whether the SEP was located in thecentral (52%), middle (24%) or lateral (24%) sectors of the 1/2blastomere. At the blastocyst stage, we de®ned the boundarybetween embryonic and abembryonic regions as a zone equivalentto 12.5% of the embryo diameter lying on either side of theblastocyst cavity roof (Fig. 4). In the main group where the SEPhad been in the central sector at the two-cell stage, 81% ofblastocysts had the SEP in this boundary zone. This compared to50% of the group where the SEP had occupied the middle sector and33% where it had been in the lateral sector at the two-cell stage. Thenumber of embryos with the SEP positioned between 1/2 blasto-meres after cleavage is completed appears to underestimate theproportion of zygotes where it is at or close to the ®rst cleavage planeduring actual division itself (as a result of blastomere rotation; seeabove). Thus, the proportion of embryos in the `central' groupcould have been initially greater than that observed here.

As the bead used to mark the SEP labelled only a small area of thefertilization cone, we sought an independent method to mark a

larger patch of the egg surface around the SEP so that we couldbetter relate this to the cleavage furrow. This was achieved byintroducing a microdrop of rhodamine-labelled concanavalin A(ConA) solution onto the egg surface. We introduced both such alabel and two ¯uorescein-labelled beads onto the fertilization coneand followed their development to the blastocyst stage in a series of15 zygotes. In all cases we found that the two markers colocalized atthe two-cell stage with the ConA extending up against the cleavagefurrow. In 13 out of 15 blastocysts, both markers were at the Em±Abborder, and in all cases beads were in the area delimited by thelabelled concanavalin (Fig. 5). Together, this shows that the SEP is astrong predictor of both the plane of ®rst cleavage and the Em±Abboundary of the blastocyst. We con®rmed this by precisely markingthe furrow separating blastomeres in two-cell stage embryos andfound that in 78% (21/27) of cases the label came to lie against theEm±Ab border.

These studies show that the SEP de®nes a plane of cleavage thatcan separate the embryo into distinct halves, and is thus animportant indicator of the future axial development of theembryo. This is an unexpected ®nding, not only because the ®rstcleavage was previously thought to orientate randomly about theA±V axis, but also because it indicates that embryonic axes becomespeci®ed at the earliest possible stage of zygotic development. Thelatter was not anticipated because early blastomeres are totipotent,allowing the embryo to regulate its development to overcomeperturbations in its organization2,8±10. However, totipotency ofboth two-cell stage blastomeres and their ability to contribute toinner cell mass (ICM) and trophectoderm lineages11 does notpreclude them from following a speci®c developmental pathway.Division of the embryo into embryonic and abembryonic hemi-spheres by the ®rst cleavage predicts a model in which descendantsof one cell would normally contribute mainly to mural trophecto-derm and ICM that will become primitive endoderm, whereas theother blastomere would develop predominantly into the polartrophectoderm and ICM destined to become epiblast (Fig. 6).This could be explained if the ®rst cleavage divided the zygoteinto two halves having different constituents, a situation for whichthere is currently no proof.

But there is an alternative possibility in which regulating thepattern of the cleavage divisions may provide a way to specifyembryonic axes that is not determinative but equips the embryowith regulative ¯exibility. It might not be the cleavage plane per sethat separates cells with differing destinies, but rather that the fate ofa particular blastomere is a direct consequence of having inheritedthe SEP. In this case the identity of each blastomere arising from the®rst cleavage would be a consequence of which one divides ®rst. Inperturbed development, we propose that regulation could beachieved if developmental processes followed the cue of whichevercell acquired such a division advantage through stochastic events.

A number of independent studies have suggested that earlier-dividing cells become preferentially incorporated into theICM7,12±14. Alteratively, early-dividing cells show a greater tendency

a b c d

Figure 5 Two markers of the egg surface colocalize from the SEP in the zygote to the

blastocyst. Zygotes were marked with beads (a, c) and ConA (b, d) at the SEP, and

examined at the two-cell (a, b) and blastocyst (c, d) stages. Two markers colocalized at

the two-cell stage with the ConA extending up against the cleavage furrow. At the

blastocyst stage, both markers are at the boundary zone between embryonic and

abembryonic regions, with the beads lying in the area delimited by the labelled ConA.

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NATURE | VOL 409 | 25 JANUARY 2001 | www.nature.com 521

to be associated with the nascent blastocoel and thus the abem-bryonic pole15. Together, the directed orientation of the initialcleavage and predisposition of the earlier dividing cells to differ-entiate ®rst may explain both their positioning on the Em±Abboundary and why the ICM and blastocoel intersect at this site.Previous lineage tracing of the fates of one out of eight cellblastomeres located either adjacent to or opposite the PB shouldhave revealed a tendency of such cells to lie on one or other side ofthe Em±Ab boundary2. Yet a substantial proportion extendedbetween these regions. This might support a model in which cellswith a division advantage have a greater role in setting identity. Butit is dif®cult to position the Em±Ab boundary by lineage tracingbecause, although clones do at ®rst stay coherent, at the later pre-implantation stages they can become disturbed in the blastocystwhen polar trophectoderm becomes recruited to mural trophecto-derm16±18. Moreover, the Em±Ab border as de®ned by the ®rstcleavage plane might not be readily seen from morphologicalfeatures.

Another model predicts that the border would not be ®xed by theposition of the blastocoel roof, but would be located parallel to itwithin the ICM showing slight variation depending whether the®rst cleavage passed on one or other side of the SEP or PB (Fig. 6).Thus, the position of the SEP and PB may determine the orientationbut not the polarity of the Em±Ab axis, the latter potentially beingsubject to the position of the SEP on either side of the ®rst cleavageplane. Indeed, it is not clear whether the blastomere inheriting theSEP has a distinct embryonic or abembryonic fate. It will be a futureobjective to understand the cellular and molecular mechanism ofthe effect exerted by the SEP, its developmental timing, and whetherit occurs at the surface or within the egg.

We conclude that two axes of the blastocyst become speci®ed inthe single-cell embryo. One of these is de®ned by the animal poleand the second, the Em±Ab axis, relates to the SEP. These axes areinitially not ®xed and can be re-established if development isperturbed2,19. Directing blastocyst organization by the plane ofcleavage and cellular identity by the order of cleavage may offer aninterpretation of the regulative events that occur following perturba-tion of development. In normal development the orientation and

timing of cleavage are mechanisms that progress hand in hand, butone might act as a failsafe mechanism for the other when develop-ment is perturbed. It will be a future challenge to determine howthese axes are initiated by the earliest events of embryogenesis andhow they become transformed into the ®nal body pattern.

MethodsCollection of eggs

Zygotes were collected from F1 (C57BL/6xCBA) females induced to superovulate1. Eggswere collected 14±15 h after hCG injection into PBS containing 200 IU ml-1 hyaluroni-dase, dispersed and then transferred to FHM plus BSA medium as described1.

Egg surface labelling and observations

Eggs were observed under an inverted (Leica) microscope using differential interferencecontrast (DIC) optics and micromanipulated with Leica micromanipulators using a DeFonbrune suction-force pump. Fluorescent (¯uorescein-labelled) beads (3±4 mm dia-meter; Polysciences) were placed in FHM (Speciality Media, Lavalette, New Jersey)medium containing 300 mg ml-1 phytohaemagglutinin for 30 min and then transferred tothe chamber containing eggs in FHM plus bovine serum albumin. Individual beads wereattached to the tip of a micropipette by suction, introduced through the zona pellucidaand placed in contact with the membrane of the fertilization cone. Once the bead hadadhered, the micropipette was withdrawn. Labelled eggs were transferred into KSOM(Speciality Media) medium and cultured as described2 to the early blastocyst stage, whenthe ratio of the blastocoel and ICM is about 1/1. In some experiments a solution of 0.5mg ml-1 of rhodamine-labelled concanavalin A (Molecular Probes) in PBS was micro-injected beneath the zona pellucida. The position of either of the markers on the embryowas scored using ¯uorescence and DIC microscopy. Live embryos were photographedusing a cooled charge-coupled-device camera (Princeton Instruments) and DIC optics ona Nikon inverted microscope.

Received 9 August; accepted 2 November 2000.

1. Weber, R. J., Pedersen, R. A., Wianny, F., Evans, M. J. & Zernicka-Goetz, M. Polarity of the mouse

embryo is anticipated before implantation. Development 126, 5591±5598 (1999).

2. Ciemerych, M. A., Mesnard, D. & Zernicka-Goetz, M. Animal and vegetal poles of the mouse egg

predict the polarity of the embryonic axis, yet are non-essential for development. Development 127,

3467±3474 (2000).

3. Gardner, R. L. The early blastocyst is bilaterally symmetrical and its axis of symmetry is aligned with

the animal±vegetal axis of the zygote in the mouse. Development 124, 289±301 (1997).

4. Goldstein, B. & Hird, S. N. Speci®cation of the anteroposterior axis in Caenorhabditis elegans.

Development 122, 1467±1474 (1996).

5. Bowerman, B. in Cell Lineage and Fate Determination (ed. Moody, S. A.) 97±117 (Academic, San

Diego, 1999).

6. Gerhart, J. et al. Cortical rotation of the Xenopus egg: consequences for the anteroposterior pattern of

embryonic dorsal development. Development 107, 37±51 (1989).

7. Graham, C. F. & Deussen, Z. A. Features of cell lineage in preimplantation mouse development.

J. Embryol. Exp. Morphol. 48, 53±72 (1978).

8. Kelly, S. J., Mulnard, J. G. & Graham, C. F. Cell division and cell allocation in early mouse

development. J. Embryol. Exp. Morphol. 48, 37±51 (1978).

9. Tarkowski, A. K. & Wroblewska, J. Development of blastomeres of mouse eggs isolated at the 4- and 8-

cell stage. J. Embryol. Exp. Morphol. 18, 155±180 (1967).

10. Rossant, J. Postimplantation development of blastomeres isolated from 4- and 8-cell mouse eggs.

J. Embryol. Exp. Morphol. 36, 283±290 (1976).

11. Balakier, H. & Pedersen, R. A. Allocation of cells to inner cell mass and trophectoderm lineages in

preimplantation mouse embryos. Dev. Biol. 23, 52±62 (1982).

12. Surani, M. A. & Barton, S. C. Spatial distribution of blastomeres is dependent on cell division order

and interactions in mouse morulae. Dev. Biol. 102, 335±343 (1984).

13. Garbutt, C. L., Johnson, M. H. & George, M. A. When and how does cell division order in¯uence cell

allocation to the inner cell mass of the mouse blastocyst? Development 100, 325±332 (1987).

14. Graham, C. F. & Lehtonen, E. Formation and consequences of cell patterns in preimplantation mouse

development. J. Embryol. Exp. Morphol. 49, 277±294 (1979).

15. Garbutt, C. L., Chisholm, J. C. & Johnson, M. H. The establishment of the embryonic±abembryonic

axis in the mouse embryo. Development 100, 125±134 (1987).

16. Copp, A. J. Interaction between inner cell mass and trophectoderm of the mouse blastocyst. II. The

fate of the polar trophectoderm. J. Embryol. Exp. Morphol. 51, 109±120 (1979).

17. Cruz, Y. P & Pedersen, R. A. Cell fate in the polar trophectoderm of mouse blastocysts as studied by

microinjection of cell lineage tracers. Dev. Biol. 112, 73±83 (1985).

18. Gardner, R. L. Clonal analysis of growth of the polar trophectoderm in the mouse. Hum. Reprod. 11,

1979±1984 (1996).

19. Zernicka-Goetz, M. Fertile offspring derived from mammalian eggs lacking either animal or vegetal

poles. Development 125, 4803±4808 (1998).

Acknowledgements

We are grateful to D. Glover for invaluable suggestions and support; R. Pedersen andC. Graham for helpful discussions; and J. Gurdon for his critical comments. This work wassupported by a Lister Institute of Preventive Medicine Fellowship, a Wellcome TrustProject Grant and a Royal Society Equipment Grant to M.Z.-G.

Correspondence and requests for materials should be addressed to M.Z.-G.(e-mail: mzg@mole.bio.cam.ac.uk).

Em EmEm

A

V VV

Zygote Two-cell embryo Blastocyst

AA

Ab Ab Ab

Figure 6 A model to show the developmental consequences of the ®rst cleavage division

de®ned by the SEP. The plane of ®rst cleavage bisects the zygote (into copper and mauve

hemispheres) passing close to the PB (green) and the SEP (yellow). The angle of this plane

can vary slightly (direction of the white arrow) depending on whether it passes on one or

other side of either the PB or the SEP (grey disc) or whether both PB and SEP together fall

on the same side of the plane (not shown). This variability is transmitted to later stages.

The corresponding position of the bead marking the SEP (yellow) is shown, together with

the distinction of the abembryonic (Ab; copper) or embryonic (Em; mauve) lineages at the

two-cell and blastocyst stages. The identity of cells could also be in¯uenced as a result of

the division advantage given to the blastomere inheriting the SEP. Although the SEP is

shown to fall on the Em side of the cleavage plane in this drawing, there is currently no

rigorous evidence to indicate a preference for either the Em or Ab side. The yellow arrows

indicate that the fate of the SEP can vary in position along the Em±Ab boundary of the

blastocyst, depending on the point at which the sperm penetrates the egg.

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