Development 99, 109-126 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

109

The formation of mesodermal tissues in the mouse embryo during

gastrulation and early organogenesis

P. P. L. TAM* and R. S. P. BEDDINGTON

Imperial Cancer Research Fund Developmental Biology Unit, University of Oxford, South Parks Road, Oxford 0X1 3 PS, UK

'Present address: Department of Anatomy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong

Summary

Orthotopic grafts of [3H]thymidine-labelled cells havebeen used to demonstrate differences in the normalfate of tissue located adjacent to and in differentregions of the primitive streak of 8th day mouseembryos developing in vitro. The posterior streakproduces predominantly extraembryonic mesoderm,while the middle portion gives rise to lateral mesodermand the anterior region generates mostly paraxialmesoderm, gut and notochord. Embryonic ectodermadjacent to the anterior part of the streak contributesmainly to paraxial mesoderm and neurectoderm. Thispattern of colonization is similar to the fate mapconstructed in primitive-streak-stage chick embryos.Similar grafts between early-somite-stage (9th day)embryos have established that the older primitive

streak continues to generate embryonic mesodermand endoderm, but ceases to make a substantialcontribution to extraembryonic mesoderm.

Orthotopic grafts and specific labelling of ecto-dermal cells with wheat germ agglutinin conjugated tocolloidal gold (WGA-Au) have been used to analysethe recruitment of cells into the paraxial mesoderm of8th and 9th day embryos. The continuous addition ofprimitive-streak-derived cells to the paraxial meso-derm is confirmed and the distribution of labelledcells along the craniocaudal sequence of somites isconsistent with some cell mixing occurring within thepresomitk mesoderm.

Key words: gastrulation, primitive streak, paraxialmesoderm, somitogenesis, mouse embryo.

Introduction

The process of gastrulation in the mouse establishesnot only the basic body plan of the animal but alsothe orderly distribution and differentiation of thedefinitive fetal tissues. Primarily, this is achieved bythe ingression, and the subsequent differentiation, ofembryonic ectoderm through the primitive streak(Snell & Stevens, 1966). That embryonic ectodermis the sole founder tissue of the fetus is strongly in-dicated by the consensus of a wide variety of studies,employing very different experimental strategies (forreview, see Beddington, 1983a). Therefore, one ofthe keys to the initial organization of the fetus, bothin terms of cellular differentiation and morpho-genetic rearrangements, must lie in the temporal andspatial pattern of tissue diversification within theembryonic ectoderm. This study extends previouswork on the fate of tissue within the embryonicectoderm (Beddington, 1981, 1982; Snow, 1981;

Copp, Roberts & Polani, 1986) by examining thefate of cells adjacent to and in different regions ofthe primitive streak in 8th day mouse embryos. Inaddition, the fate of cells from the early-somite-stageprimitive streak has been examined, in order todetermine whether or not there is a change with agein the repertoire of tissues emerging from the streak.

Morphological studies in the rat and mouse suggestthat mesoderm and definitive endoderm are theprincipal tissues produced by the primitive streak(Jolly & Ferester-Tadie, 1937; Snell & Stevens, 1966;Poelmann, 1981a,ft). In this paper, the patternof cell recruitment into mesodermal tissues hasbeen examined, using both orthotopic primitivestreak grafts of [3H]thymidine-labelled cells and byspecifically labelling the entire ectoderm populationwith wheat germ agglutinin conjugated to colloidalgold (WGA-Au) and determining the distributionof labelled cells after subsequent development inculture. The distribution of cells in the definitive

110 P. P. L. Tarn and R. S. P. Beddington

endoderm and associated primordia will be describedelsewhere (Beddington & Tam, in preparation).

The initial allocation of cells to the embryonicmesoderm has been analysed with particular refer-ence to the paraxial mesoderm. By virtue of theprecise craniocaudal segmentation of somites (Flint,Ede, Wilby & Proctor, 1978), which provides aconvenient measure of position along the embryonicaxis, the paraxial mesoderm is the most suitablemesodermal tissue in which to study the initialdeployment of cells leaving the primitive streak.There is tissue contiguity between the primitivestreak and the presomitic mesoderm, as well asbetween the streak and the gut, neural tube andnotochord, indicative of continuous cell recruitmentfrom the streak during elongation of the embryonicaxis (Snell & Stevens, 1966; Tam, 1981, Tam, Meier& Jacobson, 1982; Schoenwolf, 1984; Svajger,Kostovic-KneZevic, Bradamante & Wircher, 1985).Removal of the posterior part of the embryo, con-taining the primitive streak and/or tail bud, inter-rupts elongation of the embryonic axis and curtailssomitogenesis (Smith, 1964; Criley, 1969; Packard &Jacobson, 1976; Schoenwolf, 1977; Tam, 1986). Ifmaintenance of the paraxial mesoderm is indeeddependent upon an influx of cells from the primitivestreak (Tam, 1981; Tam & Beddington, 1986; Ooi,Sanders & Bellairs, 1986) it is of interest to knowhow these cells are distributed in the presomiticmesoderm. Morphologically discrete aggregates ofmesodermal cells, somitomeres, have been describedin the cranial and presomitic mesoderm (Tam et al.1982; Meier & Tam, 1982), and it has been shown thatthe number of somitomeres present in the presomiticmesoderm corresponds to the number of somitesgenerated by explants of the presomitic region (Tam,1986). Therefore, somitomeres may be the morpho-logical manifestation of cell allocation into metamericunits, in which case one would expect cell mixing tobe minimal within the presomitic mesoderm. Thetemporal and spatial pattern of recruitment ofprimitive-streak-derived cells into newly formedsomites has been examined in order to assess theextent of cell mixing within the presomitic mesoderm.

Materials and methods

The experimental approachTwo different experimental strategies were used to examinethe origin and initial distribution of certain mesodermaltissues during gastrulation and early organogenesis. Thefirst was to make synchronous grafts of [3H]thymidine-labelled cell clumps orthotopically into (i) different regionsof the primitive streak of 8th day embryos, (ii) into theprimitive streak of 9th day embryos and (iii) into thepresomitic mesoderm of 9th day embryos. The distribution

of labelled cells in chimaeras after 20-22 h of furtherdevelopment in vitro was analysed autoradiographically.The second approach, applied to embryos of both stages,was to label all the ectoderm cells lining the amniotic cavityby injecting a solution of wheat germ agglutinin conjugatedto colloidal gold (WGA-Au) into the cavity. This enabledthe distribution of any mesoderm which originated fromectoderm during the subsequent culture period to bedetermined. In both studies, particular attention was paidto the distribution of labelled cells in the paraxial mesoderm(the somites and presomitic mesoderm) in the expectationthat this might clarify the pattern of allocation of cells to theembryonic axis.

Recovery and culture of embryosEmbryos were obtained from a closed colony of outbredalbino PO strain (Pathology, Oxford) mice maintained on acycle of 14 h light/10 h dark, the midpoint of the dark cyclebeing 19.00h. The day on which a vaginal plug was detectedwas designated the 1st day of gestation. On the 8th and the9th day of gestation, embryos were dissected from theuterus in PB1 medium (Whittingham & Wales, 1969)containing 10 % fetal calf serum (FCS, Gibco). The parietalyolk sac was removed and the embryos were washed inseveral changes of fresh PB1+FCS medium.

After manipulation or labelling, embryos were culturedin rotating (SOrevsmin"1) 30 ml universal tubes (Sterilin)containing 3 ml of culture medium made up of equal partsof Dulbecco's modified Eagle's medium (DMEM, FlowLaboratories) and rat serum (Steele & New, 1974; New,Coppola & Cockroft, 1976; Tam & Snow, 1980). TheDMEM was supplemented with L-glutamine (58-4 mg100ml"1) and, for grafting experiments, thymidine wasadded (8X10~6M). The culture medium was sterilized byfiltration (Sartorius, pore size 0-45 ̂ m) and equilibratedwith 5 % CO2 in air overnight. Four to five embryos werecultured in each tube and at the start of culture the mediumwas gassed with a mixture of 5 % CO2, 5 % O2 and 90 % N2.Cultures were regassed after 7-8 h.

After culture (20-22 h) embryos were examined for thepresence or absence of heart beats and visceral yolk saccirculation before being transferred to phosphate-bufferedsaline (PBS). Various developmental features, such as theformation of cranial neural folds, the imagination of the gutportal and the fusion of the allantois to the chorion incultured 8th day embryos, and the closure of the cephalicneural tube, the formation of structures such as the fore-limb buds, otic capsules and pharyngeal arches, and thedegree of axial rotation in cultured 9th day embryos, werenoted. The somite number was also recorded. Grosslyabnormal and developmentally retarded embryos werediscarded. The remainder were processed for histology.After autoradiography or silver impregnation a preliminaryhistological examination was made and embryos exhibitingexcessive cell death or disorganized development wereexcluded from further analysis.

Production of in vitro chimaeras

(1) Preparation of labelled graftsFollowing the removal of the parietal yolk sac, 8th dayembryos were transferred to medium containing the alpha

Mesoderm formation in the mouse embryo 111

modification of Eagle's medium (Flow Laboratories)supplemented with 30 /JM each of adenosine, guano-sine, cytidine and uridine, 10% FCS and 10/iCiml"1

of [3H]thymidine (specific activity 12-5 CimM"1; Radio-chemicals, Amersham). The embryos were labelled for2h under conditions described previously (Beddington,1981). 9th day embryos were labelled for 3h in rotatingtubes containing rat serum + DMEM + 10/iCiml"1 [3H]-thymidine (specific activity 12-5CimM"1).

After labelling, embryos were washed for 10-15 min inthree changes of PB1 + FCS containing an additional8X10~6M of thymidine. Some embryos were then fixed inCarnoy's fluid to serve as controls for the degree of[3H]thymidine incorporation (uptake controls). In everyexperiment, some labelled embryos (labelled controls) werecultured for the same duration as experimental embryos.Labelled controls serve to ensure the compatibility of[3H] thymidine incorporation with normal development andalso provide an important measure of the expected dilutionof label in different tissues during the culture period(Beddington, 1981).

The remaining labelled embryos were dissected withsiliconized (Repelcote) fine glass needles to isolate theembryonic ectoderm required for grafting. Fig. 1 illustratesthe four regions (B, C, D and E) dissected for orthotopic

B

Fig. 1. A diagram of the right half of an 8th dayprimitive-streak-stage embryo showing the sites oforthotopic grafting of [3H]thymidine-labelled cells. A is aregion at the anterior extreme of the primitive streakwhich corresponds to the 'node' region. B (anteriorstreak) lies in the anterior third portion and C(midstreak) corresponds to the middle portion of thestreak. D (posterior streak) is at the posterior end of thestreak close to the base of the allantoic bud (al). E marksan area of the embryonic ectoderm immediately lateral tothe anterior region of the streak. Although area E ismarked on the right side of the embryo, orthotopic gTaftswere always made to a similar area on the left side of theembryo.

Fig. 2. A longitudinal section through the fragment oftissue isolated from an 8th day embryo containing theprimitive streak and the 'node' region. The area fromwhich cells were isolated for grafting is marked withboxes and these correspond to the regions marked inFig. 1. The epithelial (ectodermal) portion of theprimitive streak was always used in the graftingexperiments described in this study. The arrow pointscranially. Haematoxylin and eosin (H&E). Bar, 50^m.Fig. 3. A transverse section through the midregion (C) offragment containing the primitive streak. The dashedlines demarcate the region of primitive streak from whichclumps of cells were isolated for grafting. H&E. Bar,50 urn.

grafting in 8th day embryos. Region A, corresponding tothe most anterior extreme of the primitive streak, rep-resents the area receiving orthotopic and heterotopic graftsin previous experiments (Beddington, 1982) and is includedbecause colonization of,the paraxial mesoderm in thesechimaeras has been further analysed in this study. Themechanical isolation of the tissue for grafting (Figs 2, 3) wassimilar to that described elsewhere (Beddington, 1982), thefinal graft used for injection containing approximately15-30 cells.

In order to isolate the primitive streak fragment from the9th day embryo, the posterior region of the embryo wasfirst separated from the trunk by a transverse cut made atthe level of the last-formed somite. A wedge of tissue wasobtained from the posterior region of the neuropore bymaking two oblique cuts as shown in Fig. 4. The primitivestreak tissue was then isolated by a horizontal cut throughthe fragment (Fig. 5), followed by further dissection toremove as much of the adhering mesenchyme as possible(Fig. 6). Presomitic mesoderm was mechanically isolated

112 P. P. L. Tarn and R. S. P. Beddington

Fig. 4. A diagram of the posterior region (dorsal view) ofthe 9th day early-somite-stage embryo showing theposition of two oblique cuts made medial to the neuralfolds (nf) at the posterior neuropore in order to isolate awedge of tissue containing the primitive streak. This wasfurther dissected as shown in Fig. 5. The box on thelateral side of the embryo marks the region from whichlabelled presomitic mesoderm tissue was obtained fororthotopic grafting.

from the region shown in Fig. 4. The potential graft tissuewas further subdivided with needles into clumps of asuitable size for injection, each consisting of about 40 cells(see below).

(2) Grafting of labelled tissuesThe developmental stage of the recipient embryos wasalways matched as closely as possible with that of donorsto minimize any asynchrony between graft and host.Primitive-streak-stage embryos were matched according tosize and certain morphological features such as the extent ofamnion formation and the appearance of an allantoic bud.Somite number in conjunction with the extent of cephalicneurulation (Jacobson & Tarn, 1982) were used to compare9th day embryos.

The clumps of labelled cells and the recipient embryoswere transferred to a drop of PB1+FCS containing8X10~6M of thymidine. The manipulation of the 8th dayembryos was carried out in a hanging drop of the medium ina Leitz manipulation chamber filled with liquid paraffin(Boots UK Ltd) mounted on the fixed stage of a Zeiss(Ergoval) binocular microscope. 9th day embryos wereinjected in a drop of the medium located on the lid of aculture dish (Sterilin) and covered with paraffin. Thepreparation of holding and injection pipettes and thebasic grafting procedure have been described before(Beddington, 1981). In more advanced 9th day embryos(6-8 somites), it was not possible to directly observe theinjection pipette in the primitive streak. The pipette wasdirected through the primitive streak via the hindgut portalso that its tip protruded from the posterior region of the

Fig. 5. An autoradiograph of the posterior region of a9th day embryo after labelling for 3h with [3H]thymidine.Virtually all cells in the streak and the underlyingmesenchyme are labelled. The dashed line indicates theplane of a horizontal cut that is made to obtain afragment of the primitive streak. Arrow points cranially.H&E. Bar, 50/an.Fig. 6. A transverse section of an explant of the primitivestreak of a 9th day embryo. The fragment contains theepithelial (ectodermal) portion of the streak and somemesenchyme found at the basal aspect of the streak. Thedashed lines demarcate the region from which clumps ofcells were isolated for grafting. H&E. Bar, 50/im.

embryo into the amniotic cavity. The pipette was thenslowly withdrawn and the graft expelled when its tipdisappeared into embryonic tissue.

Fig. 7 shows a clump of labelled cells in the primitivestreak region of a 9th day embryo fixed immediately aftergrafting. Most of the graft is located in the deep aspect ofthe primitive streak and is in intimate contact with theoverlying endoderm. In a series of fifteen 9th day embryoswhich were examined autoradiographically immediatelyafter grafting, a mean number of 38-7 (±4-8) cells werefound. In most cases, the graft was located in the midlinebut in four the labelled clump was found slightly (approxi-mately 100 fan) to one side of the streak.

Autoradiography

The preparation of autoradiographs and the criteria usedfor identifying and counting colonizing donor cells were thesame as those described by Beddington (1981).

Mesoderm formation in the mouse embryo 113

Lectin-conjugate-labelling experimentsPreparation of the WGA-Au label was as describedby Horisberger & Rosset (1977) and Smits-van Prooije,Poelmann, Dubbeldam, Mentink & Vermeij-Keers (1986).The WGA molecules (Sigma) were first cross linked tobovine serum albumin (BSA, Miles Laboratory) by re-acting with glutaraldehyde. The WGA-BSA complex wasthen conjugated in the presence of polyethylene glycol tocolloidal gold particles [10-15 nm; 0-005% solution incitrate buffer pH5-5 (Polysciences)]. The WGA-BSA-Auconjugate was spun at 120 000 g for 30min at 4-10°Cin a Beckman L5-50 ultracentrifuge. The pellet was re-suspended in PBS solution A (Oxoid), recentrifuged andwashed three times. A concentrated WGA-Au label wasobtained by suspending the final pellet in about 50/il ofcitrate buffer pH5-5. The final preparation has an equiv-alent of 1-2 mg WGA mP1 and has a shelf life of at least a

month if kept at 4°C. The concentration of free WGA usedfor injection was 1 mg ml"1 in PBS. Injection of free WGAwas undertaken to eliminate the possibility of spurioussilver impregnation (see below) in the presence of lectinalone.

The apparatus and procedure for injecting a smallquantity of the label into the aminotic cavity were similar tothose for grafting of labelled cells. Prior to injection, themicropipettes were calibrated with an ocular micrometer onthe microforge so that the volume of solution delivered wasstandardized to about 0-2 nl (for 8th day) and 10 nl (for 9thday) for injection of free WGA. A larger volume of about2nl (for 8th day) and 10 nl (for 9th day) of WGA-Au labelwas injected. Based on the estimation by Burgoyne, Tarn &Evans (1983), the 8th day embryo contains about 15 nl fluidin the amniotic cavity, whereas the volume of amniotic fluidin the 9th day embryo is about 500 nl (Cockroft, personalcommunication). To reduce the risk of forced diffusion of

Fig. 7. An autoradiograph of a 9th day embryo fixed immediately after grafting of [3H]thymidine-labelled cells. In thisembryo, about 43 cells were placed in the primitive streak. The indentation in the endoderm (en) is caused by thepassage of the micropipette during the course of grafting, al, allantois. H&E. Bar, 100/im.Fig. 8. A longitudinal section through the somite (im) of a chimaeric embryo produced by orthotopic grafting ofprimitive streak cells to a 9th day embryo. Both the dermamyotome and the sclerotome are colonized by labelled cells(arrowheads). H&E. Bar, 50ism.Fig. 9. A transverse section of the presomitic mesodenn (psm) of a chimaeric embryo produced by orthotopic graftingof labelled primitive streak cells to a 9th day embryo. Arrowheads mark a group of labelled cells in the dorsolateralregion of the presomitic mesodenn. H&E. Bar, 50/im.

114 P. P. L. Tarn and R. S. P. Beddington

the label through the ectodermal epithelium caused byexcessive hydrostatic pressure, injection of label wasusually preceded by the withdrawal of an equivalent volumeof the amniotic cavity fluid from the embryo using a secondmicropipette (internal diameter 5-10 fim). This pipette wasmounted adjacent to the injection pipette. The injectionpipette was always inserted through the visceral yolk sacand the amnion to prevent damage to the embryonicregion. After trimming the ectoplacental cone, for identifi-cation purposes, uninjected embryos (unlabelled controls)were placed in the manipulation drop during the injectionof operated embryos and were subsequently cultured in thesame tube as labelled embryos.

Embryos injected with WGA-Au or free WGA and theunlabelled controls were fixed in Carnoy fluid, embedded inparaffin wax and serially sectioned at 8jan. Gold particleswere visualized by a silver enhancement procedure (Snow& Springall, personal communication). The dewaxed andhydrated sections were treated with a silver developer(2-35 g trisodium citrate dihydrate (BDH), 2-55 g citric acid(BDH), 0-85g hydroquinone (BDH) and O l lg silverlactate (Fluka) dissolved in 100 ml milli-Q water) at pH3-9for 5-7 min, followed by fixing in a photographic fixer(Unifix, Kodak). The treatment resulted in the formation ofsilver grains of 100-200 nm around the gold particles whichcan be readily seen by light microscopy. The sections werecounterstained with 0-25 % aqueous solution of fast greenfor better contrast of the silver grains. Analysis of unlabelledcontrols revealed that individual silver grains were occasion-ally present in cells but that the occurrence of two or moresilver grains in a single cell was extremely rare. Cells ininjected embryos were regarded as positively labelled whentwo or more silver grains were found embedded in theircytoplasm.

Results

Embryonic development in vitroOver 80 % of 8th day unlabelled control embryos andlabelled controls developed normally during 20-22 h inculture (Table 1). They formed 4-5 pairs of somitesand showed no significant difference from embryos of

the same age recovered from in vivo with respectto the various morphological features assessed(Table 1). Similarly, embryos receiving [3H]thy-midine grafts or those injected with WGA-Au or freeWGA showed development comparable to controls,although those labelled with lectin or lectin-conjugatehad a slightly elevated somite number (Table 1). The9th day early-somite-stage embryos also underwentextensive growth and morphogenesis in culture(Table 2). Again, over 80% of labelled controls,grafted and injected embryos developed normallyand showed no consistent deviation from the patternof development seen in unlabelled and in vivo controls(Table 2). A somewhat reduced incidence of axialrotation was observed in WGA-Au-injected em-bryos and of those labelled with unconjugated WGAonly 50 % developed a visceral yolk sac circulation(Table 2). It is unlikely that these minor anomalieswere a specific result of lectin binding becauseWGA-Au- and WGA-injected embryos differed inthe particular characteristics affected.

The results in Tables 1 and 2 show that, in general,development of 8th and 9th day embryos was notadversely affected by the labels used or by grafting,and that the extent of differentiation and morpho-genesis permitted a detailed analysis of the normalfate of grafted or labelled tissue. Specific assessmentof embryonic growth, by measurement of total pro-tein content, was not undertaken because previousstudies using similar culture conditions have estab-lished that growth during the first 24 h in vitroparallels that in vivo (Beddington, 1981; Tarn &Snow, 1980; Tarn, 1986). Most embryos appearednormal histologically although approximately 10 % ofboth control and experimental 8th day embryos wereexcluded from further analysis due to a markeddeficiency of cranial mesenchyme and severely atten-uated neural epithelium. Excessive cellular necrosiswas seen in two macroscopically normal 9th day

Table 1. The development of 8th day embryos in vitro following grafting, labelling and culture

In vitro chimaerasLabelled controlGrafted

Lectin-conjugate labellingControlWGA-goldWGA

In vivo 9th day embryo(3 litters)

Significantly different from

No. ofembryos

1158

2456

619

No.developednormally

9 (82 %)46 (79 %)

21 (88 %)44 (79 %)6 (100 %)

16 (84 %)

Neuralfolds

9(100)46(100)

21 (100)40(91)6(100)

15(94)

No. (%) of embryos with

Gut

8(89)42 (91)

15 (71)37(84)6(100)

13 (81)

control at *, P< 0-001; t, P < 0 0 5 by Student's unpaired t-

Heartbeat

7(78)32 (70)

19(90)34(71)4(67)

15(94)

test.

Fusedallantois

7(78)36(78)

21 (100)38(86)6(100)

12 (75)

Somiteno. (n)

51 ±0-6 (9)5-0 ± 0-3 (41)

4-2 ±0-5 (18)6-2 ± 0-3 (42)*6-3±0-2(6)t5-0 ±0-3 (16)

Mesoderm formation in the mouse embryo 115

Table 2. The development of 9th day embryos following grafting, labelling and culture

In vitro chimaerasLabelled controlGrafted

Lectin-conjugate labellingControlWGA-goldWGA

In vivo 10th day embryos(3 litters)

No. ofembryos

2673

4677

821

Initialsomite no.

(n)

4-5 ±0-3 (22)5-1 ±0-2 (44)

4-7 ± 0-2 (38)4-5 ± 0-3 (57)5-8 ±0-3 (8)

—

Significantly different from the control at *, /><005

No.embryos

developednormally

24 (92 %)61 (84 %)

35 (76 %)65 (84 %)8 (100 %)

18 (86 %)

; t, P<0-01

Yolk saccirculation

23(96)49(80)

29(83)49 (75)4(50)*

17(94)

by chi-square

No. (%)

Neural tubeclosure

16 (61)42(69)

16(46)49 (75)t3(38)

10(56)

: test.

of embryos

Beatingheart

24 (100)56(92)

33(94)58(89)8(100)

18 (100)

showing

Forelimbbuds

13(54)38(62)

20(57)30(46)5(63)

12 (67)

Axisrotation

23(96)52(85)

29(83)38 (58)*6(75)

16(89)

Somiteno. (n)

18-6 ±0-8 (22)18-0 ± 0-5 (58)

16-9 ± 0-7 (35)16-8 + 0-5(65)19-0 ±1-0 (8)17-2 ±0-5 (18)

embryos that had received grafts, and these were alsoexcluded from further analysis.

The distribution of [3H]thymidine-labelled cells inchimaeras

(1) Uptake and labelled controls

After 2 h in labelling medium dense grains were seenover all nuclei visible in every tissue of the primitive-streak-stage embryos (see Beddington, 1981). Theprimitive streak region of 9th day embryos appearedto be similarly labelled following incubation in[3H]thymidine for 3h (Fig. 5). The density of label-ling in other tissues of 9th day uptake controls wasmore variable but over 95 % of nuclei were labelled.After 20-22 h in culture silver grains could still bedetected over at least 90 % of nuclei in 8th and 9thday labelled controls. In cultured 8th day embryosgut endoderm, notochord and paraxial mesodermexhibited the highest density of grains, whereasneurectoderm and surface ectoderm appeared leastlabelled. Labelling was equally widespread but gener-ally less dense in 9th day labelled controls. Nuclei ingut endoderm, notochord and cardiac mesodermwere more heavily labelled than those in the cranialmesenchyme or brain. The spinal cord, paraxialmesoderm and primitive streak exhibited a morepatchy distribution of both densely and lightlylabelled nuclei. Both stages of labelled controls hadfairly uniformly labelled visceral yolk sac endodermwhilst the mesodermal component showed morevariable levels of labelling. These results indicate thatall the cells in a graft would be labelled and that most,if not all, of their progeny should be detectableautoradiographically after 20-22 h of further develop-ment in culture.

(2) Orthotopic grafts to 8th day embryosTable 3 shows the distribution of [3H]thymidine-labelled cells in chimaeric embryos obtained from

orthotopic grafts to regions B,C,D and E (Fig. 1). Of46 embryos analysed, 35 proved to be chimaeric(76 %). Grafts to the posterior extreme of the streak(D) generated chimaerism predominantly in extra-embryonic mesoderm (mesoderm of the amnion,visceral yolk sac and allantois). There was no contri-bution to paraxial mesoderm but some to lateralmesoderm (here defined as that embryonic meso-derm lateral to the paraxial mesoderm). Labelledtissue injected into the middle of the streak (C)colonized almost exclusively lateral mesoderm. Moreanterior grafts (B) contributed to paraxial mesoderm,the primitive streak, notochord and head process/notochordal plate. In addition, two of the chimaeraswere colonized entirely in the cranial mesenchyme.Grafts lateral to the anterior part of the streak (E)gave rise largely to paraxial mesoderm althoughcolonization of other mesodermal tissues was ob-served and three embryos were chimaeric in theneural tube. In these chimaeras, labelled cells wereusually found on the same side of the embryo as theoriginal graft (left side) but in four cases somelabelled cells were also located contralaterally. Ortho-topic grafts to all regions produced some chimaerasthat were colonized in the primitive streak itself.

In the above series, chimaerism in the paraxialmesoderm was almost always confined to the preso-mitic mesoderm, incorporation into definitive somitesprobably requiring a longer period of development inculture. In a previous study (Beddington, 1981,1982),where embryos were cultured for 36 h, orthotopic andheterotopic grafts of posterior streak tissue to theanterior extreme of the primitive streak (A) gave riseto chimaerism in definitive somites as well as in thepresomitic mesoderm. From 20 orthotopic and 23heterotopic grafts 25 chimaeras were obtained. Ofthese, 10 were chimaeric in the paraxial mesoderm.The distribution of labelled cells along the cranio-caudal axis of these chimaeras has now been mapped

Tab

le 3

. T

he d

istr

ibut

ion

of [

3HJt

hym

idin

e-la

bell

ed

cell

s in

em

bryo

nic

tiss

ues

foll

owin

g or

thot

opic

gra

ftin

g in

the

8th

da

y em

bry

o a

nd

cu

ltu

red

fo

r 2

0-2

2 h

2 3 50 to I 1

Post

enor

str

eak

(D)

Chi

mae

ras

23

45

67

89

10

Mid

stre

ak (

C)

12

3 4

5

Ant

erio

r st

reak

(B

)

12

3 4

5 6

7

Lat

eral

-ant

erio

r ec

tode

rm (

£)

12

34

56

78

9 10

11

12

Neu

ral

tube

Surf

ace

ecto

derm

Cra

nial

mes

ench

yme

End

othe

lium

Para

xial

mes

oder

mL

ater

al m

esod

erm

Prim

itive

str

eak

+ca

udal

mes

ench

yme

Not

ocho

rdH

ead

proc

ess/

noto

chor

dal

plat

e

Gut

end

oder

m

Ext

raem

bryo

nic

mes

oder

mA

mni

onV

isce

ral

yolk

sac

Alla

ntoi

s

No.

of

labe

lled

cells

1 5

1 4

1281

21

32

20

13

5

14

1 [6

81

2 30

1

9

1 1

2

4 7

4 [j

9j|

18

5 9

11

3 n

Ji

2

4 |8

8|| 1

5 I

2 10 17

4 12

4 17

6

2 5

3 18

[5]

][54

] 1

15 [

IF]

4 38

[79]

27

26 3

4

36

137

128

206

118

44

138

93

94

4835

25

10

8 30

7

81

28

28

25

25

17

21

2931

30

3

36

17

38

130

20

79

7 9

7

| |

deno

tes

the

tissu

e in

whi

ch S

40 %

of

the

labe

lled

popu

latio

n w

as f

ound

.

Mesoderm formation in the mouse embryo 117

and is shown in Fig. 10. The most anterior cells arefound in the myelencephalon, which corresponds tothe seventh somitomere of Meier & Tam (1982).Approximately 50-80 % of the chimaeras havelabelled cells in somites caudal to the third somite andthey are all chimaeric in the presomitic mesoderm.The number of labelled cells in each somite wasusually not more than two and not all the somites in agiven chimaera were colonized. Labelled cells werefound in somites on both sides of the embryo but onlyin 35 % of cases (29/82) were both somites at thesame level colonized. Four chimaeras had labelledcells in the primitive streak. No difference in thepattern of distribution was seen between embryosreceiving orthotopic and heterotopic grafts.

(3) Orthotopic grafts to 9th day embryosAutoradiographs were prepared from 61 9th dayembryos that had received grafts in the primitivestreak region. 41 (67 %) were found to be chimaeric.Table 4 shows the distribution of labelled cells in 26 of

100-1

80-

§60'

40-|

20-

0-1 11iv v vi VII 2

Cranialsomitomeres

6 8 10 12 14 16 18 psmSomites

Fig. 10. The distribution of [3H]thymidine-labelled cellsto the paraxial mesoderm of chimaeras produced bygrafting primitive streak cells to the 'node' region of the8th day embryo followed by 36 h of culture. Thesechimaeras are part of a collection described byBeddington (1981, 1982). The arrows mark the stage ofdevelopment of the embryo, at the time of grafting, interms of the metameric units formed in the paraxialmesoderm, which correspond to the 4th and 5thsomitomeres in the cranial mesenchyme at the late-primitive-streak stage. A total of 10 chimaeras wereanalysed and the histogram shows the percentage ofchimaeras having a labelled somite at various segmentallevels of the axis. It can be seen that only somitomeres orsomites formed after grafting are colonized and that thepresomitic mesoderm is always chimaeric. Because of thelonger period of culture, these chimaeras formed moresomites than those described in Table 3. A somite isconsidered as labelled if it contains one labelled cell,psm, presomitic mesoderm.

these chimaeras. The pattern of colonization in theremaining 15 embryos was similar but, to simplify thepresentation of results, only embryos with more than30 colonizing cells are included in Table 4.

The labelled cells in these chimaeras were mainlydistributed to the paraxial mesoderm (Figs 8, 9), theprimitive streak and the lateral mesoderm. Labelledcells were also found in the endoderm of the hindgutand the neighbouring notochord (Table 4). Themajority of the labelled cells were found in the trunkand caudal regions of the embryo. The only cranialcontribution was the presence of four labelled cells inthe cranial mesenchyme (chimaeras 1, 3 and 18).Probably, as a result of the route adopted for grafting,unincorporated clumps of labelled cells were often(13/26) seen in the hindgut lumen.

Fig. 11 shows the distribution of labelled cells inthe somites that were formed after grafting. It isapparent that the more posterior somites are the onesmost frequently colonized and contain the mostlabelled cells. In some chimaeras labelled somiteswere confined to one side of the primitive streak. Inseven chimaeras labelled cells were found in the firstfew somites formed immediately after the graft wasinjected. This occurred despite the fact that the initiallocation of the graft was at least three or four somitelengths away from the region of somite segmentation(see Tam, 1986). This might be accounted for by ananterior movement of cells in the presomitic meso-derm prior to somite segmentation. However, suchmovement might be overestimated if manipulation ofthe embryo caused a delay in somitogenesis.

Cell mixing was examined more directly by ortho-topically grafting a clump of about 40 presomiticmesoderm cells. In all four of the resultant chim-aeras, obtained from transplanting cells to 10embryos, three or four contiguous somites werecolonized in an otherwise nonchimaeric sequence ofsomites (Table 5). Labelled cells were found inter-spersed with host cells both in the sclerotome andin the dermamyotome of colonized somites. Thissuggests that some cell mixing must have occurredwithin the presomitic mesoderm because it is highlyunlikely that donor cells could invade the epithelialcomponent of a somite after segmentation has takenplace. Very rarely, colonization of more anteriorsomites, which were already segmented at the time ofgrafting, was also seen: labelled cells were found inone somite in five chimaeras and in two somites in onechimaera. Each of these somites was colonized byonly one or two labelled cells (see Discussion).

The distribution of WGA-Au-labelled cellsInjection of WGA-Au into the amniotic cavityresulted in extensive labelling of the embryonicectoderm of 8th day embryos (Fig. 12). A similar

118 P. P. L. Tarn and R. S. P. Beddington

Table 4. The distribution of [3HJthymidine-labelled primitive streak cells to embryonic tissue after graftingorthotopically on the 9th day and cultured for 20-22 h in vitro

Chimaeras (n = 26)

Tissues 1 2 3 4 5 8 9 10 12 14 15 17 18 22 23 24 28 29 30 32 33 35 37 38 40 41

Neural tubeSurface ectoderm

Cranial mesenchymeHeart + endotheliumParaxial mesodermLateral mesodermPrimitive streak

Gut endodermNotochord

Extraembryonic mesodenn

Total no. of labelled cells

| 3911

1

3 3 3 3 8 2 2

1

2 2 4

1 2 1 1

18 [l5l][«[] 12 8 3 [82][20][45][49j 12 [32|| 29 || 18 || 15 fl 94 || 23 | 10 13

6 65 17 1 34 21 2 [IT] 2 3 3 10 8 [27][25

5 48 36 14 1151 10 11 10 1 2 [351 1 17 1 8 8 8

4 26 9 |_45j 10 |_57j 13 4 10

1 110 16 1 5 12 17

3 3 117

5 1 2 5

73 231 183 92 57 108 131 36 118 53 78 47 35 50 33 99 48 60 69 70 89 45 91 58 149 330

I I denotes the tissue that was colonized by &40 % of the labelled population.

1001

0-10 2 4 6 8 10 12 14 16

Somites formed after graftingpsm

Fig. 11. The distribution of labelled cells to the paraxialmesoderm of the chimaeric embryos produced byorthotopic grafting of [3H]thymidine-labelled cells to theprimitive streak on the 9th day embryos followed by20-22h of culture. Because of the variation in the initialsomite number of the embryos (3-7 pain of somites), thesomites formed after grafting were scored in eachchimaera in a numerical order starting from the last-formed somite at the time of grafting (somite 0, markedby arrow). Somites 1 to 16 are therefore formedsubsequently in culture and those to the left of the arroware already segmented before the experiment. Altogether41 chimaeras were scored. The histogram shows thepercentage of embryos having a labelled somite at varioussegmental levels of the body axis. A somite is consideredlabelled if it contains one labelled cell, psm, presomiticmesodenn.

blanket labelling of the ectodermal tissue and theprimitive streak was also achieved in 9th day embryos(Fig. 14). Embryos cultured for 3h after injectionshowed positively labelled cells deep in the primitivestreak (Figs 13, 14) but there was no sign of silver-enhanced gold particles elsewhere in the mesodermor underlying endoderm. Uninjected embryos andembryos injected with free WGA did not containsilver deposits characteristic of gold particle labelling.

After 20-22h in culture, 8th and 9th day embryosinjected with WGA-Au contained numerous labelledcells in the neurectoderm and surface ectoderm. Inaddition, some cranial mesenchyme was labelled: inearly-somite-stage embryos, derived from cultured8th day embryos, labelled cells were located in thelateral subectodermal region, whereas cultured 9thday embryos had positively labelled cells not only inthis region but also in the mesenchyme of the pharyn-geal arches and in cellular condensations at theprospective sites of cranial nerve ganglia. Theselabelled cells may have been of neural crest orplacodal origin. The cranial mesenchyme around thebase of the neural tube and notochord contained few,if any, labelled cells.

The distribution of gold-containing cells in theparaxial mesoderm is shown in Fig. 15. The pattern issimilar to that obtained with [3H]thymidine-labelledgrafts: those somites or regions of cranial mesen-chyme formed after the start of labelling were fre-quently labelled and the presomitic mesoderm andprimitive streak almost invariably contained positivecells. Again, labelled cells were found in somites

Mesoderm formation in the mouse embryo 119

Table 5. The distribution of [3 HJthymidine-labelled presomitic mesoderm cells to somites following orthotopicgrafting to the presomitic mesoderm of 9th day embryos

(1)(2)(3)(4)

Embryos

Initial Finalsomite no. somite no.

6 205 145 185 14

Somite 6

o

o

o

o

Somite 7

0500

No.

Somite 8

0400

of [3H]thymidine-labelled cells in

Somite 9

0222

Somite

12015

10 Somite 11

28042

Somite 12

180

170

Somite 13

0000

formed immediately after the injection of label and ina very few cases somites present at the time ofinjection were also labelled. Anterior somites wereonly considered to be unequivocally labelled if morethan 5% (approximately 5-10 cells) of the dermo-myotome contained gold particles. The sclerotomewas ignored because of the possibility of scoringmigrating neural crest cells. Examples of labelledtissues are shown in Figs 16-18.

In most embryos the number of cells containinggold particles was of the order of 5-15 per somite,20-30 in the presomitic mesoderm and about 30 inthe lateral mesoderm. Undoubtedly, if WGA-Auwere behaving as a truly heritable marker, one wouldexpect to see more positive cells. Furthermore, de-spite ubiquitous labelling of ectoderm cells initially,some negative cells were seen in the neuroepitheliumand surface ectoderm after development in culture.However, the absence of obviously extracellular goldparticles suggests that the relatively low number oflabelled cells is probably due to unequal partition ofparticles at cell division rather than to the loss of labelfrom the cytoplasm. Nonetheless, it is important torecognize that in this experiment only a fraction ofthe progeny of initially labelled cells is being ident-ified.

Discussion

Both the developmental fate of cells in the primitivestreak and the pattern of cell allocation within theparaxial mesoderm of intact late-primitive-streak-and early-somite-stage mouse embryos have beenexamined. Compelling evidence has been obtainedfor regionalization in the deployment of cells atdifferent levels of the primitive streak during the laterstages of gastrulation. However, the analysis of thedistribution of cells in the presomitic mesoderm andtheir recruitment into somites proved somewhat lessconclusive.

Regionalization of cell fate

Previous studies in the mouse have indicated thatcells invaginating at different points along the streak

may have distinct developmental fates. When specificfragments of the streak are isolated in vitro, withoutdisturbing the relationship of the constituent germlayers, a difference is seen between different regionsin their propensity to form allantois, tail bud struc-tures and primordial germ cells (Snow, 1981). Ortho-topic grafts, similar to those employed in this study,have demonstrated that the anterior extreme ofthe primitive streak, corresponding in location toHensen's node in the avian embryo (Snell & Stevens,1966; Rugh, 1968; Bellairs, 1986), generates gutendoderm and notochord, as suggested by severalmorphological studies (Jolly & Ferester-Tadie, 1936;Jurand, 1974; Poelmann, 1981a; Tam & Meier, 1982),in addition to lateral and paraxial mesoderm andvascular endothelium (Beddington, 1981). Grafts tothe posterior part of the streak, on the other hand,produce exclusively mesodermal tissues (Beddington,1982). In accordance with isolation experiments(Snow, 1981) and histochemical observations(Ozdzenski, 1967), primordial germ cells are alsoproduced by orthotopic grafts to this region (Coppet al. 1986). The current study extends the map of theprimitive streak and confirms that different pro-spective mesodermal tissues emerge from differentregions of the streak (Table 3). Furthermore, grafts toearly-somite-stage embryos demonstrate that theprimitive streak remains an active source of meso-dermal tissue at least during the early stages oforganogenesis (Table 4). This is consistent with thewide variety of differentiated tissues observed inexperimental teratomas derived from ectopic graftsof caudal 9th day tissues containing the primitivestreak (Tam, 1984). The primitive streak may alsoremain regionalized, in terms of different develop-mental fates, in the 9th day embryo but the bulkinessof the posterior region and diminished size of thestreak precluded accurate localization of grafts alongits length at this stage.

The putative origin of the various embryonic andextraembryonic tissues along the craniocaudal axis ofthe 8th day primitive streak, and the predominantcontribution to paraxial mesoderm by embryonicectoderm located lateral to the anterior part of the

120 P. P. L. Tarn and R. S. P. Beddington

streak, is remarkably similar to the situation de-scribed for the chick embryo at a comparable devel-opmental stage (stages 3-4; Pasteels, 1937; Spratt,1955; Rosenquist, 1966; Nicolet, 1970, 1971; Vakaet,

1984). Thus, in both chick and mouse embryos, cellsemerging from the anterior part of the streak contrib-ute mainly to gut, notochord and paraxial mesoderm.Cells from the middle region give rise to lateral

14

mesoderm, whereas the caudal part of the streak isdevoted largely to the provision of cells for extra-embryonic mesoderm. Interestingly, whereas graftsto the early-somite-stage primitive streak continue tocolonize definitive embryonic fetal tissues, there wasminimal chimaerism in the extraembryonic meso-derm suggesting that recruitment of cells into thistissue ceases during early organogenesis.

In all categories of graft, after 20-22 h some chim-aeras retained labelled cells in the primitive streak(Tables 3,4) . These cells were interspersed with hostcells, showed the same dilution of label as equivalentcells in labelled controls and did not resemble un-incorporated graft tissue. This suggests that the streakitself may act as a source of cells and not just as aroute for relocation. Certainly, an elevated mitoticindex is found in the primitive streak throughoutearly organogenesis (Tarn & Beddington, 1986),which would be consistent with the notion that itgenerates cells to supplement the prospective popu-lations passing through it. A somewhat similar func-tion for the primitive streak has been proposed in thechick embryo, where experiments involving extir-pation and grafting of the streak indicate that thepresomitic mesoderm has a dual origin: one popu-lation coming from the presumptive somitic meso-derm and the other derived from a continual influxof cells migrating away from the regressing streak(Bellairs & Veini, 1984; Bellairs, 1985; Ooi etal.1986).

In the chick embryo, using labels such as vital dyes,carbon particles and [3H]thymidine or grafting quailtissue, it has been shown that epiblast cells lateral to

Figs 12, 13. 8th day primitive-streak-stage embryoslabelled with WGA-gold conjugate by intra-amnioticinjection and examined after 3 h of labelling. TheWGA-Au label is found in the embryonic ectoderm (e)of the embryo (Fig. 12). No leakage has occurred and thelabel is confined to the ectodermal side of the amnion{am). At higher magnification (Fig. 13), the WGA-Aulabel is seen in the deeper aspect of the primitive streak(ps) and occasionally in the mesoderm at the streak(arrowheads) but not in the endoderm. Silver enhancedand counterstained with fast green. Bar, 50^m.Fig. 14. 9th day early-somite-stage embryos labelled withWGA-gold conjugates by intra-amniotic injection andexamined after 3 h of labelling. The conjugate labels theentire neurectoderm (NE) and the primitive streak regionof the 4-somite-stage embryo. Label is not found in themesoderm of the cranial region or in the paraxialmesoderm but some mesenchymal cells beneath thestreak are labelled. Only the ectoderm of the amnion{am) is labelled. Some cells at the base of the allantois(a/) are also labelled with the conjugate, fg, foregut; sm,somite. Silver enhanced and fast-green stained. Bar,100/an.

Mesoderm formation in the mouse embryo 121

100-i A

SO'

§.«)•

40-

20' J l

ill iv vCranial

somitomeres

2 4 6 8 10 psmSomites

100

80-

to

.oE

1 40-

20-

0-4 6 8 10 12 14 16Somites formed after labelling

18 psm

Fig. 15. The distribution of WGA-Au-labelled cells inthe paraxial mesoderm of embryos labelled on (A) the8th day and (B) the 9th day, followed by 20-22 h ofculture. The arrows indicate the stage of development ofembryos at the beginning of labelling. At this stage, the8th day embryo has formed the 4th or 5th somitomeres inthe cranial mesenchyme. For the 9th day embryo, thearrow marks the last-formed somite (somite 3 to 7) in theparaxial mesoderm at the commencement of labelling,with reference to which somites formed subsequently arescored. A total of 33 8th day embryos and 47 9th dayembryos were studied. In these embryos, somites areclassified as labelled if they contain more than five cellswith silver grains. The cranial somitomeres (I to VII) areidentified by their topographical relation to the brainparts and are classified as labelled when silver-grain-bearing cells are found in the mesenchyme at the base ofthe brain and in the perinotochordal mesenchyme. ThehistogTams show the percentage of embryos havinglabelled metameric units at various segmental levels ofthe embryonic axis, psm, presomitic mesoderm.

122 P. P. L. Tarn and R. S. P. Beddington

the anterior part of the streak represent the presump-tive somitic mesoderm (Gallera, 1975; Nicolet, 1971;Vakaet, 1984). These cells converge towards thestreak and become situated along its anteriormargins as the streak starts to regress. At the sametime the prospective neurectoderm cells move cau-dally to flank the prospective paraxial mesoderm (see

Nicolet, 1971). If similar morphogenetic movementsare occurring in the mouse this would account for thecolonization patterns seen in grafts to region E(Fig. 1; Table 3), where contribution to paraxialmesoderm and to neurectoderm are observed. Thecolonization of presomitic mesoderm in grafts to theanterior streak (region B) also mimics the situation in

en

16

•psm

18

Fig. 16. The labelled neural plate (np), primitive streak (ps) and presomitic mesoderm {psm) of an embryo labelledwith WGA-Au on the 8th day and cultured for 20-22h. en, endoderm. Examples of labelled cells are marked byarrowheads. Silver enhanced and fast-green stained. Bar, 50/an.Fig. 17. The posterior region of an embryo labelled with WGA-Au on the 9th day showing labelling in the neural plate(np) and the presomitic mesoderm {psm). The prospective lateral mesoderm (Im) is also labelled. Only a few labelledcells are seen in the hindgut endoderm (hg). Silver enhanced and fast-green stained. Examples of labelled cells aremarked by arrowheads. Bar, 50/on.Fig. 18. The labelled somites (s) and presomitic mesoderm {psm) of an embryo labelled with WGA-Au on the 9th day.se, surface ectoderm. Examples of labelled cells are marked by arrowheads. Silver enhanced and fast-green stained.Bar, 50 fan.

Mesoderm formation in the mouse embryo 123

the chick. The chimaerism in cranial mesenchymeobserved in two embryos receiving grafts in region Bis consistent with the experimental evidence thatcranial somitomeres are located in the vicinity of theHensen's node in the chick at stages 3-4 (Meier &Jacobson, 1982). Cranial somitomeres give rise tocranial mesenchyme (Meier & Tarn, 1982), whichmay be considered as an integral part of the paraxialmesoderm of the embryo. It has been proposed thatthe cranial somitomeric mesoderm emerges from theprimitive streak immediately before that of thesomites. The contribution of cells to the cranialmesenchyme by grafts to region B but not by those toregion E lends some support to this notion. In otherwords, cells located in the anterior streak, whichinvaginate before those lying lateral to the streak, arethose giving rise to cranial mesenchyme in this series.These results, therefore, are compatible with the ideathat paraxial mesoderm emerges from the streak instrict temporal order corresponding to its final cranio-caudal location.

It is unlikely that the regionalization found in theprimitive streak reflects prior commitment of theembryonic ectoderm to form different kinds of meso-dermal tissues. Heterotopic grafts of both cranial andcaudal regions of the streak into the anterior partof the embryo result in colonization of definitiveectodermal derivatives (Beddington, 1982) and intro-duction of prospective definitive ectoderm into thecaudal end of the streak produces chimaerism in em-bryonic and extraembryonic mesoderm (Beddington,1982; Copp etal. 1986). In addition, most regionsof the embryonic ectoderm appear pluripotent inectopic grafts (Beddington, 19836; Chan & Tarn,1986; Svajger, Levak-Svajger & Skreb, 1986). There-fore it seems probable that the emergence of distinc-tive mesodermal populations from different regionsof the streak may relate more to morphogeneticconstraints on cell movements through and awayfrom the streak, distributing hitherto uncommittedcells to different parts of the embryo where sub-sequent tissues interactions determined their event-ual differentiation, than to any intrinsic differences inthe developmental potential of the cells themselves.Alternatively, the streak itself could be acting as anorganizing centre in the sense that as cells passthrough it they become committed to a particulardevelopmental pathway (Tarn, 1981; Tarn & Bedding-ton, 1986).

Cell recruitment into the paraxial mesodermThe pattern of cell recruitment into paraxial meso-derm was analysed both in in vitro chimaeras and byobserving the contribution of initially ectodermalcells, collectively labelled with WGA-Au, to the fileof somites and presomitic mesoderm. Injection of

WGA-Au resulted in extensive and specific labellingof the embryonic ectoderm (8th day), neurectodermand surface ectoderm (9th day), amniotic ectodermand the superficial cells of the primitive streak. Therewas no evidence, even after short-term culture, fornonspecific transfer of gold particles to other tissuelayers. This corresponds to the pattern of labellingreported previously for conjugated and free WGA inembryos of a similar stage (Gesink, Poelmann,Smits-van Prooije & Vermeij-Keers, 1983; Smits-vanProoije etal. 1986; Tan & Morriss-Kay, 1986). How-ever, it is probable, due to unequal partition of goldparticles, or aggregates, during cytokinesis, that onlya sample of the progeny of this labelled populationcan be detected after prolonged culture. The likeli-hood of spurious negative cells precludes the use ofthis method to determine whether or not presomiticmesoderm comprises two populations: a residentpopulation, which is supplemented by an influx ofcells from the streak (Bellairs, 1985). However, thesequence of recruitment of labelled cells into somites,in both chimaeras and WGA-Au-labelled embryos,should reflect the behaviour of cells within the pre-somitic mesoderm, with respect to the existence orotherwise of a stable metameric prepattern. In otherwords, if the somitomeric pattern apparent in thepresomitic mesoderm (Tarn etal. 1982; Tarn, 1986) isa morphological manifestation of a stable segregationof mesodermal cells into prospective somites, onewould not expect to see labelled cells appearing insomites derived from those somitomeres present atthe time of labelling.

The pattern of [3H]thymidine-labelled donor cellcolonization and the appearance of WGA-Au-labelled cells in somites is basically similar. In both8th and 9th day embryos the most posterior somites,presomitic mesoderm and primitive streak arelabelled or contain donor cells. This supports thenotion that the paraxial mesoderm is continuallysupplemented with cells emerging from the streak.Primitive-streak-stage embryos, at the time of injec-tion, would be expected to have four to five somito-meres (Tarn & Meier, 1982). The most anteriorlocation of colonization donor cells is in the myelen-cephalic mesenchyme (Fig. 10) which is thought to bederived from the seventh somitomere (Meier & Tam,1982). WGA-Au labelling resulted in a few chim-aeras containing labelled cells anterior to the my-elencephalon but the majority were labelled caudal tothe first somite (Fig. 15). Therefore, in general, theredoes not appear to be much mixing within the earliestpopulation of paraxial mesoderm cells.

The results from 9th day embryos are somewhatconfusing, although the general impression gained isof cell mixing within the presomitic mesoderm super-imposed on a net caudocranial movement of cells

124 P. P. L. Tarn and R. S. P. Beddington

through this tissue. Precise localization of grafts ismore difficult at this stage and, therefore, someatypical patterns of colonization are inevitable. Forexample, in six chimaeras, donor cells were identifiedin a total of seven somites that had already formed atthe time of grafting. Colonization of pre-existingsomites was almost invariably restricted to the sclero-tome and may represent intermingling between dis-persing sclerotomal cells. Alternatively, a few cellsmay have been introduced directly into the anteriorregion during grafting. In 2 (out of 15) embryos fixedimmediately after grafting about five donor cells wereseen in the cranial mesenchyme, suggesting that theinjection pipette may have been pushed too farthrough the primitive streak and consequently pen-etrated the anterior region. It is also possible thatcells inadvertently deposited in the amniotic cavitymay find their way into the embryo in a manneranalogous to that of injected neural crest cells(Jaenisch, 1985).

Despite these few anomalous results, in essenceboth the in vitro chimaeras and the WGA-Au-labelled 9th day embryos present a similar picture. Atthe time of manipulation the presomitic mesodermwould be expected to contain six distinct somitomeres(Tam et al. 1982). Nonetheless, progeny of graftedtissues and cells containing gold particles were foundin somites that formed immediately after grafting orlabelling, although the incidence of colonization orlabelling increased in somites formed later (Figs 11,15). The most plausible explanation for the coloniz-ation of somites formed immediately after manipu-lation is that there may be extensive cell mixingwithin the presomitic mesoderm, such that cellsentering from the primitive streak are rapidly dis-tributed along the length of the presomitic meso-derm. However, this mixing probably coincides witha net caudocranial movement of the total populationbecause grafts of presomitic mesoderm producedonly three or four chimaeric somites in the middle ofan otherwise nonchimaeric sequence of somites andno donor cells were found in the presomitic meso-derm or primitive streak. Both the ultrastructure ofsomitomeres (Tam et al. 1982) and the absence ofcomplex junctions between mesodermal cells (Flint &Ede, 1978) would be compatible with cell mixingwithin the presomitic mesoderm, although such mix-ing argues against somitomeres being self-containedprecursors of somites.

We thank Professor Richard Gardner, Dr Michael Snow,Dr David Cockroft and Dr Andy Copp for their helpfuldiscussion and advice, and Mrs Jo Williamson for preparingthe manuscript. P.P.L.T. is supported by a CroucherFoundation Fellowship and R.S.P.B. by a Fellowship fromthe Lister Institute of Preventive Medicine.

References

BATTEN, B. E. & HAAR, J. L. (1979). Fine structuraldifferentiation of germ layers in the mouse at the timeof mesoderm formation. Anal. Rec. 194, 125-142.

BEDDINGTON, R. S. P. (1981). An autoradiographicanalysis of the potency of embryonic ectoderm in the8th day postimplantation mouse embryo. J. Embryol.exp. Morph. 64, 87-104.

BEDDINGTON, R. S. P. (1982). An autoradiographicanalysis of tissue potency in different regions of theembryonic ectoderm during gastrulation in the mouse.J. Embryol. exp. Morph. 69, 265-285.

BEDDINGTON, R. S. P. (1983a). The origin of the foetaltissues during gastrulation in the rodent. InDevelopment in Mammals, vol. 5 (ed. M. H. Johnson),pp. 1-32. Amsterdam: Elsevier.

BEDDINGTON, R. S. P. (1983b). Histogenetic andneoplastic potential of different regions of the mouseembryonic egg cylinder. /. Embryol. exp. Morph. 75,189-204.

BELLAJRS, R. (1985). A new theory about somiteformation in the chick. In Developmental Mechanisms:Normal and Abnormal, pp. 25—44. New York: Alan R.Liss Inc.

BELLAJRS, R. (1986). The primitive streak. Anat.Embryol. 174, 1-14.

BELLAIRS, R. & VEINI, M. (1984). Experimental analysisof control mechanisms in somite segmentation in avianembryos. II. Reduction of material in the gastrulastages of the chick. J. Embryol. exp. Morph. 79,183-200.

BURGOYNE, P. S., TAM, P. P. L. & EVANS, E. P. (1983).Retarded development of XO conceptuses duringpregnancy in the mouse. J. Reprod. Fert. 68, 387-393.

CHAN, W. Y. & TAM, P. P. L. (1986). The histogeneticpotential of neural plate cells of early-somite-stagemouse embryos. /. Embryol. exp. Morph. 96, 183-193.

COPP, A. J., ROBERTS, H. M. & POLANI, P. E. (1986).Chimaerism of primordial germ cells in the earlypostimplantation mouse embryo followingmicrosurgical grafting of posterior primitive streak cellsin vitro. J. Embryol. exp. Morph. 95, 95-115.

CRILEY, B. (1969). Analysis of the embryonic sources andmechanisms of development of posterior levels of chickneural tube. /. Morph. 128, 465-502.

FLINT, O. P. & EDE, D. A. (1978). Cell interactions inthe developing somite: in vivo comparisons betweenamputated (am/am) and normal mouse embryos. J. CellSci. 31, 275-291.

FLINT, O. P., EDE, D. A., WILBY, O. K. & PROCTOR, J.(1978). Control of somite number in normal andamputated mouse embryos: an experimental and atheoretical analysis. /. Embryol. exp. Morph. 45,189-202.

GALLERA, J. (1975). At what stage of development doesthe somitic mesoblast invaginate into the primitivestreak of the chick embryo? Experientia 31, 584-585.

GESINK, A. F., POELMANN, R. E., SMTTS-VAN PROOUE, A.E. & VERMEU-KEERS, CHR. (1983). The cell surfacecoat during closure of the neural tube, as revealed by

Mesoderm formation in the mouse embryo 125

concanavalin A and wheat germ agglutinin. J. Anat.137, 418-419.

HORISBERGER, M. & ROSSET, J. (1977). Colloidal gold, auseful marker for transmission and scanningmicroscopy. J. Histochem. Cytochem. 25, 295-305.

JACOBSON, A. G. & TAM, P. P. L. (1982). Cephalicneurulation in the mouse embryo analyzed by SEMand morphometry. Anat. Rec. 203, 375-3%.

JAENISCH, R. (1985). Mammalian neural crest cellsparticipate in normal embryonic development onmicroinjection into post-implantation mouse embryos.Nature, Lond. 318, 181-183.

JOLLY, J. & FERESTER-TADIE, M. (1936). Recherches surl'oeuf du rat et de la souris. Archs Anat. microsc.Morph. exp. 32, 323-390.

JURAND, A. (1974). Some aspects of the development ofnotochord in mouse embryos. /. Embryol. exp. Morph.32, 1-33.

MEIER, S. & JACOBSON, A. G. (1982). Experimentalstudies of the origin and expression of metamericpattern in the chick embryo. J. exp. Zool. 219,217-232.

MEIER, S. & TAM, P. P. L. (1982). Metameric patterndevelopment in the embryonic axis of the mouse. I.Differentiation of the cranial segments. Differentiation21, 95-108.

NEW, D. A. T., COPPOLA, P. T. & COCKROFT, D. (1976).Comparison of growth in vitro and in vivo ofpostimplantation rat embryos. J. Embryol. exp. Morph.36, 133-144.

NICOLET, G. (1970). Analyse autoradiographique de lalocalisation des diff6rentes 6bauches pr6somptives dansla ligne primitive de l'embryon de Poulet. /. Embryol.exp. Morph. 23, 79-108.

NICOLET, G. (1971). Avian gastrulation. Adv. Morph. 9,231-262.

Ooi, E. C. V., SANDERS, E. J. & BELLAIRS, R. (1986).The contribution of the primitive streak to the somitesin the avian embryo. J. Embryol. exp. Morph. 92,193-206.

OzDZENSKf, W. (1967). Observations on the origin ofprimordial germ cells in the mouse. Zool. Pol. 17,367-379.

PACKARD, D. S. JR & JACOBSON, A. G. (1976). Theinfluence of axial structures on chick somite formation.Devi Biol. 53, 36-48.

PASTEELS, J. (1937). Etudes sur la gastrulation desvertdbre's meroblastiques. III. Oiseaux. IV.Conclusions generales. Archs Biol. 48, 381-488.

POELMANN, R. E. (1981a). The formation of theembryonic mesoderm in the early post-implantationmouse embryos. Anat. Embryol. 162, 29-40.

POELMANN, R. E. (19816). The head process and theformation of the definitive endoderm in the mouseembryo. Anat. Embryol. 162, 41-49.

ROSENQUIST, G. (1966). A radioautographic study oflabelled grafts in the chick blastoderm. Development

from primitive streak stage to stage 12. Contr. Embryol.Carneg. last. 38, 71-110.

RUGH, R. (1968). TTie Mouse. Minneapolis: Burgess.SCHOENWOLF, G. C. (1977). Tail (end) bud contributions

to the posterior region of the chick embryo. J. exp.Zool. 201, 227-246.

SCHOENWOLF, G. C. (1984). Histological andultrastructural studies of the secondary neurulation inmouse embryos. Am. J. Anat. 169, 361-376.

SMITH, L. J. (1964). The effects of transection andextirpation on axis formation and elongation in theyoung mouse embryo. /. Embryol. exp. Morph. 12,787-803.

SMTTS-VAN PROOUE, A. E., POELMANN, R. E.,

DUBBELDAM, J. A., MENTINK, M. M. T. & VERMELJ-KEERS, CHR. (1986). WGA-Au as a novel marker formesectoderm formation in mouse embryos cultured invitro. Stain Tech. 61, 97-106.

SNELL, G. D. & STEVENS, L. C. (1966). Earlyembryology. In Biology of the Laboratory Mouse (ed. E.L. Green), pp. 205-245. New York: Dover Pub. Inc.

SNOW, M. H. L. (1981). Autonomous development ofparts isolated from primitive-streak-stage mouseembryos. Is development clonal? J. Embryol. exp.Morph. 65 Supplement, 269-287.

SPRATT, N. T. JR (1955). Analysis of the organizer centerof the early chick embryo. Localization of prospectivenotochord and somitic cells. J. exp. Zool. 128, 121-164.

STEELE, C. E. & NEW, D. A. T. (1974). Serum variantscausing the formation of double hearts and otherabnormalities in explanted rat embryos. J. Embryol.exp. Morph. 31, 707-719.

SVAJGER, A., KOSTOVN5-KNE2EVIC', L., BRADAMANTE, 2. &

WIRCHER, M. (1985). Tail gut formation in the ratembryo. Wilhelm Roux Arch, devl Biol. 194, 429-432.

SVAJGER, A., LEVAK-SVAJGER, B. & SKREB, N. (1986).Review article. Rat embryonic ectoderm as renalisograft. /. Embryol. exp. Morph. 94, 1-27.

TAM, P. P. L. (1981). The control of somitogenesis inmouse embryos. J. Embryol. exp. Morph. 65Supplement, 103-128.

TAM, P. P. L. (1984). The histogenetic capacity of tissuesin the caudal end of the embryonic axis of the mouse.J. Embryol. exp. Morph. 82, 253-266.

TAM, P. P. L. (1986). A study on the pattern ofprospective somites in the presomitic mesoderm ofmouse embryos. J. Embryol. exp. Morph. 92, 269-285.

TAM, P. P. L. & BEDDINGTON, R. S. P. (1986). Themetameric organization of the presomitic mesodermand somite specification in the mouse embryo. InSomites in Developing Embryos (ed. R. Bellairs, D. A.Ede & J. Lash). New York: Plenum Pub. Co. (inpress).

TAM, P. P. L. & MEIER, S. (1982). The establishment of asomitomeric pattern in the mesoderm of thegastrulating mouse embryo. Am. J. Anat. 164, 209-225.

126 P. P. L. Tam and R. S. P. Beddington

TAM, P. P. L., MEIER, S. & JACOBSON, A. G. (1982).Differentiation of the metameric pattern in theembryonic axis of the mouse. II. Somitomericorganization of the presomitic mesoderm.Differentiation 21, 109-122.

TAM, P. P. L. & SNOW, M. H. L. (1980). The in vitroculture of primitive-streak-stage mouse embryos./. Embryol. exp. Morph. 59, 131-143.

TAN, S. S. & MORRISS-KAY, G. (1986). Analysis of cranialneural crest cell migration and early fates in

postimplantation rat chimaeras. J. Embryol. exp.Morph. 98, 21-58.

VAKAET, L. (1984). Early development of birds. InChimaeras in Developmental Biology (ed. N. LeDouarin& A. McLaren), pp. 71-88. London: Academic Press.

WHimNGHAM, D. G. & WALES, R. G. (1969). Storage oftwo-cell mouse embryos in vitro. Austr. J. biol. Sci. 22,1065-1068.

(Accepted 8 October 1986)