J. Anat. (1986), 145, pp. 35-47 35With 13 figuresPrinted in Great Britain

Regional and temporal differences in the parietalendoderm of the midgestation mouse embryo

D. L. COCKROFT

Sir William Dunn School ofPathology, South Parks Road,Oxford, OX1 3RE, U.K.

(Accepted 13 June 1985)

INTRODUCTION

The midgestation mouse fetus is surrounded by the parietal yolk sac whichconsists of parietal endoderm (PE) cells, their basement (Reichert's) membrane andtrophoblastic giant cells. The PE cells are apposed to the visceral endoderm internallyand are separated from the trophoblast and its surrounding decidua externally byReichert's membrane. Unlike the visceral endoderm cells, which form a continuousepithelial sheet, the parietal endoderm cells are arranged as a loose network, withoutextensive cell contacts (Wislocki & Padykula, 1953; Jollie, 1968; Smith & Strickland,1981). The parietal endoderm/Reichert's membrane complex (PE/R) is in contin-uity with visceral endoderm at its insertion into the base of the placenta, and inmidgestation it assumes a spherical or ellipsoid surface.Although there has been much recent interest in the PE as a model system for

studying basement membrane synthesis and composition (Minor et al. 1976a, b;Hogan, 1980; Hogan, Cooper & Kurkinen, 1980), very little is known about itsnormal development in the postimplantation embryo. Because of the relativedifficulty of isolating the PE/R complex close to the placenta, most previousobservations and studies have been made on the capsular parietal endoderm (i.e.the part distal to the placenta); hence the possibility exists that there may be regionaldifferences between proximal (placental) and distal parietal endoderm, perhapscorrelated with differences in proliferative and biosynthetic activity. This paperdescribes the results of light and scanning lelectron microscopical and time-lapsestudies, which show that the morphology of PE cells in the placental region differsmarkedly from that of distal PE cells, and that placental PE cells also show greatermotile activity than their distal counterparts.

MATERIALS AND METHODS

Mice of the Pathology Oxford strain were killed at stages between the ninth andeleventh days of pregnancy (day of plug taken as first day of pregnancy). The uteriwere removed and torn open in phosphate-buffered saline, and the conceptusesseparated and transferred to PB1 medium (Whittingham & Wales, 1969) containingglucose (1 g/l) instead of lactate, and fetal calf serum (10 % v/v) in place of bovineserum albumin, where first the decidua and then the trophoblast cells were removed,with watchmakers' forceps. The trophoblast cells adhered quite firmly to Reichert'smembrane, but with care they could be peeled off as a sheet, leaving cleanedReichert's membrane as the outermost covering of the conceptus. Using iridectomyscissors, the PE/R complex and visceral endoderm were cut in half in a plane

parallel to the distal/proximal (relative to the placenta) axis, and the incision wasextended proximally so as also to divide the placenta transversely. The fetus, visceralyolk sac and most of the placenta were then separated and discarded, leaving twoproximodistal halves of the PE/R complex, each with a small piece of placentaattached. These fragments of the PE/R complex were then stuck to pieces of trans-parent plastic, cut from Falcon plastic dishes, to which the edges of the membraneadhered when they were pressed into it with the tips of watchmakers' forceps(Gardner, 1984). The fragments of placental tissue were located in indentations inthe plastic, drilled to a depth of 0.5 mm with a 1 5 mm bit mounted in a pillar drill,allowing the PE/R complex to lie flat on the plastic, cells uppermost. This wasunnecessary with ninth day membranes, where little placental tissue was involved.The preparations were fixed in formol saline, stained with Giemsa, and mounted

in Aquamount (BDH, England). The distribution of smooth and blebby cells in thesepreparations, shown in Figure 5, was determined using a Zeiss RA microscope fittedwith a drawing tube to trace successive microscope fields, each 85 x 233 ,um(18955 tm2), radiating out from the placenta to the distal tip, and the number ofeach cell type in each field was scored. The majority of cells could readily be assignedto one or other category; for the remainder (2-5 %) an arbitrary criterion wasadopted: that cells with two or more blebs were classified as blebby, while theremainder were categorised as smooth.

Scanning electron microscopy (SEM)Pieces of PE/R complex from ninth to fourteenth day fetuses were obtained as

before and attached to pieces of plastic measuring 6 x 12 mm. The larger mem-branes were cut into proximal, middle and distal pieces of more manageable size. Thepreparations were fixed for I hour at 4°C in 2 5 % glutaraldehyde in 0 1 M cacodylatebuffer, postfixed for 1 hour in 2 % osmium tetroxide, dehydrated, critical point driedwith carbon dioxide in a Polaron drying apparatus, sputter coated with gold, andexamined with a JEOL JEM 100 CX transmission/scanning electron microscope.

Time-lapse recordingFresh preparations from tenth and eleventh day embryos were made as described

above, the PE/R complex being attached to pieces of plastic measuring 12 x 25 mm.Each preparation was placed in a 50 ml flat sided tissue culture flask (Falcon)containing 10 ml of a-medium supplemented with 10 % fetal calf serum, with a dabof silicon grease on each corner of the plastic to hold it in place on the bottom of theflask. The air in the flask was replaced with a gas mixture consisting of 5 % carbondioxide 95 % air, and the flask was placed on the stage of a Leitz Diavert invertedoptics microscope housed in a 37 °C room. The microscope was fitted with a NationalPanasonic WV1850B video camera connected to a (remote) NEC PVC 9507 U-matic time-lapse video recorder. After a period of 2 hours to allow equilibration ofthe system and recovery of the cells from the explantation procedure, recording wascommenced at a speed which corresponded to a 64 fold increase in speed (comparedwith real time) on playback. Each membrane was video taped for at least 30 minutesand was arranged with the placenta at one edge of the field, and more distal regionsat the other edge. At the end of recording, the preparations were fixed in formolsaline, stained with Giemsa and mounted in Aquamount. The total period of culturein each case was 6 hours.The video recordings were assessed independently by three observers. The monitor

36 D. L. COCKROFT

Mouse parietal endoderm1001 .

0)~~~~~~~~~~~~~~~~1> 50

.0

a)\

1 2 3 4 5

Field number

Proximal Distal

Fig. 1. Proportion of blebby versus smooth cells in successive microscope fields of freshlyisolated ninth day parietal endoderms. Each line links the data from one preparation.

100

..........V ... ...3...

.50 -.6

0Art1 2 3 4 5 6 7 8 9

Field number

Proximal Distal

Fig. 2. Proportion ofblebby versus smooth cells in successive microscope fields of freshly isolatedtenth day parietal endoderms. Each line links the data from one preparation.

screen was divided by a vertical line and each half of the parietal endoderm, placental(proximal) and (relatively) distal, was rated for cell activity on a scale of 0-3 (Osignifying negligible activity, 3 denoting very vigorous activity). The scores for eachfield were averaged.

RESULTS

Distribution ofsmooth and blebby cellsThe appearance of proximal and distal regions of the parietal endoderm in the

Giemsa-stained preparations used to. analyse regional variations in cell morphologyis shown in Figure 5. Figures 1-3 and,Table 1 show the distribution of smooth and

37

100 l

0> 50,0

....

1 3 5 7 9 1 1 13 15 17

Field number

Proximal Distal

Fig. 3. Proportion of blebby versus smooth cells in successive microscope fields of freshlyisolated eleventh day parietal endoderms. Each line links the data from one preparation.

100

50 o

- X 'oX \

ORA -

..... ......**~.*0 0

1 2 6 7 8 9 10Field number

Proximal Distal

Fig. 4. Proportion of blebby versus smooth cells in successive microscope fields of parietalendoderms isolated on the tenth day and cultured for 6 hours. Each line links the data fromone preparation.

blebby cells in freshly isolated preparations from mouse embryos on the ninth, tenthand eleventh day of gestation respectively.With the sole exception of one (the smallest) of the ninth day membranes, the

distribution of the two morphological classes of cell was distinctly non-random, with80-90 % of the cells proximal to the placenta exhibiting the blebby phenotype, whilein distal regions the majority of the cells were relatively smooth surfaced. Chi-squared tests were performed on the number of cells of each type in each field for allthe scored fields on each membrane, and the results for all but one of the prepara-tions were highly significant (P <0001) for each membrane. The exception was thesmallest ninth day membrane mentioned above, on which nearly all the cells were

38 D. L. COCKROFT

Mouse parietal endoderm

Table 1. Distribution of smooth and blebby cells in freshly isolated parietal yolk sacs(PEIR complex) ofmouse embryos on the ninth, tenth and eleventh day ofgestation

Minimum x% blebby % blebby X2 for Average maximum diameter

cells cells Number of 50 % whole number of of intactin first in last countable point membrane cells/ PE/R complex

Membrane field* fieldt fieldsl (fields)§ (D.F.) II field (mm)

Ninth day A 94 94 3 0 595 (2)B 88 0 5 3-4 217-2(4)C 93 64 4 37-9 (3)D 94 75 3 14-1(2)E 83 57 4 16-3 (3)F 88 49 5 5 0 70-8 (4)

Ninth dayaverages 90 57 4-0 4.3

Tenth day G 96 24 6 5-1 164-0(5)H 90 46 7 6-4 67 5(6)I 88 15 9 5-2 266-4 (8)J 83 1 7 4X8 241X8 (6)K 78 35 6 4X1 59-6 (5)L 79 7 7 3-7 204-8 (6)M 69 1 9 1P8 2926(8)N 84 38 8 6'8 133-2(7)

Tenth dayaverages 83 21 7-4 4-7

Eleventh day 0 89 5 15 6-8 8025 (14)P 77 13 17 2-5 122-8 (8)Q 94 7 13 8-5 306-8 (6)R 84 2 17 4 5 342 5 (8)

11082841048474

0 8 x1P21P2 x 1P310 x 1P30O9x 1-11POx 1P4P-Ox 1P5

90 lOx13

83 2-1x22594 2-3x28116 2-9x 3-189 28x3 1

106 2-3x27102 2-9x 3-285 2-5x3 089 2-9x 3-1

96 2-6 x 2-9

114 3 9x44989 31x3-9104 3 5x440100 4-0x50

Eleventh dayaverages 86 7 15-5 5 6 - 102 3-6x445* Field nearest placenta.t Field furthest from placenta.t Indicates the distance from the placenta of the last field and is a rough measure of the size of the

membrane. All fields were scored on membranes A-0; on membranes P, Q and R alternate fields werescored.

§ Intersection of graph of % blebby cells versus fields with 50 % line (see Figs. 1-3).11 x2 for membrane A is not significant; all others are significant with P< 0-001. D.F. = degrees of

freedom.

blebby, and their distribution was not different from random. Although the distribu-tion pattern, i.e. decreasing numbers of blebby cells with increasing distance fromthe placenta, was clear from the figures, it was noticeable that there was considerablevariation in the distribution of these cells, even in preparations of the same age.Nevertheless, Table 1 shows that there were also some clear age-related differences.The most obvious was the proportion of blebby cells in the most distal field, whichdecreased from an average of 57 % in the ninth day membranes, to only 7 % ineleventh day membranes. This appeared to be mainly a consequence of the size(length) of the membranes, and this was reinforced by the data on 50 % pointsshown in Table 1. Although the size (length) of the membranes, as indicated by thenumber of fields, was roughly doubling with each day of development, the distancefrom the placenta to the 50 % points (where half the cells were blebby and half were

39

D. L. COCKROFT

Table 2. Distribution of blebby (versus smooth) cells in the parietal yolk sacs (PEIRcomplex) of tenth day mouse embryos after 6 hours in culture

Minimum x% blebby % blebby x' for Average maximumdiameter

cells cells Number of 50 % whole number of of intactin first in last countable point membrane cells/ PE/R complex

Membrane field* fieldt fieldst (fields)§ (D.F.)UI field (mm)

S 74 9 7 2-3 183-1(6) 97 2-4 x 2-7T 79 3 6 1-8 238-6(5) 94 2 6 x 2'7U 91 7 10 2-9 331-7(9) 92 2-9 x 3'2V 66 14 9 1-7 83-3 (8) 85 2'6 x 3 0

Averages 78 8 8.0 2-2 - 92 2-6 x 2-9* Field nearest placenta.t Field furthest from placenta.t Indicates the distance from the placenta of the last field and is a rough measure of the size of the

membrane. All intermediate fields were scored.§ Intersection ofgraph of % blebby cells versus fields with 50 % line (see Fig. 4).11 x' for each membrane is significant withP<0 001.

D.F. = degrees of freedom.

smooth) changed only slightly with age, implying that the spatial distribution of thetwo cell types between the placenta and this point did not change greatly over theage range studied. It should also be noted that the proportion of blebby cells in fourof the ninth day preparations never fell as low as 50 %, and those two membranesin which it did were the two largest of this age.

Figure 4 and Table 2 show the distribution of blebby cells in tenth day membranesthat had been cultured for 6 hours after explantation. Comparison with the data forfreshly fixed tenth day preparations showed that there was a general reduction in theproportion of blebby cells in the cultured membranes, particularly in the distalregions. Associated with this was a marked reduction in the distance from theplacenta to the 50 % point, from an average of 4-7 fields to 2-2 fields. This differencewas significant (P < 0 02) using Student's t-test.

Scanning electron microscopyThe spatial distribution of smooth and blebby cells described above was observed

at all ages, butwith the greater resolution of the scanning electron microscope, furtherage-related differences became apparent.Ninth day membranes, as suggested previously, showed a preponderance of

blebby cells in all regions (Fig. 6a, b). The cells in Figure 6b appear to be confluent,a phenomenon which was occasionally but by -no means universally observed inninth and tenth day specimens. The younger (ninth and tenth day) embryos, in theextreme proximal region of the parietal endoderm, had large rounded cells withnumerous short filopodia, some of which also exhibited blebs (Fig. 7). Such cellswere not in evidence in this region in the more advanced embryos.

Preparations from eleventh day embryos displayed relatively smooth cells distallywhilst retaining the blebby phenotype proximally (Fig. 9a, b). The distal cells andalso those in the middle regions of preparations from more advanced embryos weresmoother still (Figs. lOb, llb) while the proximal cells (Figs. lOa, 1la) weresimilar to those of younger embryos. In the middle and distal regions of thepreparations from the oldest (thirteenth and fourteenth day) embryos studied

40

Mouse parietal endoderm 41

Giems. (a)Proxial reion, b) dital rgion x540proima rein ajinin th plcna Tecls hvnueoshrt filooi admsalsob

exhbi blbs x,3200....

~~~~~~~~~~~(.Fig. 8. Light icrograph of tenthdaybparietal endoderm after 6 hours in culture duringwhicha

number of clumps of cells have appeared (arrows). xI270.

FAGiesa (a Prxia rein'b' dita region...x 540.

Fig. 5(-b.Scningheetromicrographs of tenth day parietal endodermpreparations sthiedextrempima aroximal region,adoiin theplceta.Thegin cel haenmeossor40.odaadmotas

rexhiitn beshoni b huhteresnmru.x 3200.

Fig. 8. Light micrograph of tenth day parietal endoderm after 6 hours in culture during which anumber ofclumps of cells have appeared (arrows). x 270.

D. L. COCKROFTin' I ..

4-

A

(a) 9 (b) W s AFY

10(b) 1~~~~~~~~~~~0

Fig. 9(a-b). Scanning electron micrographs of eleventh day parietal endoderm cells on Reich-ert's membrane. The proximal cells in (a) are blebby, while the distal cells (b) are relativelysmooth and rounded. x 3200.Fig. 10(a-b). Scanning electron micrographs of twelfth day parietal endoderm cells on Reich-ert's membrane. The proximal cells in (a) exhibit blebs, while the distal cells in (b) are extremelysmooth surfaced. (a) x 4100; (b) x 4900.Fig. 11 (a-b). Scanning electron micrographs of thirteenth day parietal endoderm cells onReichert's membrane. The proximal cells in (a) have the same blebby phenotype found in thisregion in younger embryos, while those in the middle shown in (b) and distal regions aresmooth and rounded, with variable numbers of short filopodia. x 3200.

42

.L

Mouse parietal endoderm

Fig. 12(a-b). Scanning electron micrographs of middle region fourteenth day (a) and thirteenthday (b) parietal endoderm cells on Reichert's membrane. Most of the cells have numerous shortfilopodia and some ofthem are very large. x 2400.Fig. 13(a-b). Scanning electron micrographs of the middle regions of tenth shown in (a) andtwelfth shown in (b) day parietal endoderm, showing rounded and dividing cells. (a) x 2400;(b) x4100.

were cells with numerous short filopodia; some of these cells were very large andflattened (Fig. 12a, b). These filopodial cells were quite similar in appearance tothose found in the extreme proximal region in the younger embryos (Fig. 7) though,in the older embryos, blebs were never seen on them. No cells with such filopodiawere found in eleventh or twelfth day parietal endoderms. In all but the oldestembryos, rounded and dividing cells could be found in both proximal and distalregions of the parietal endoderm (Fig. 13 a, b).Time-lapse recordingFourteen tenth day and thirteen eleventh day preparations were assessed for cell

movement in the placental and distal regions of the parietal endoderm. All of thetenth day preparations, and all but one of the eleventh day preparations showedgreater activity in placental than in more distal regions. When the scores for eachregion were averaged (Table 3) the greater activity of placental parietal endodermwas highly significant (P < 0-001 for each age). Furthermore, there was a decline incell motility in each region with increasing age (Table 3). Although very vigorous

43

.10 ..t

12 a 12 (b)

NAD> , V..

D. L. COCKROFT

Table 3. Motile activity ofparietal endoderm cells in different regions of tenth andeleventh day membranes

Placental Distal Placental/Number of activity* activity* distal

Age of membrane membranes ± S.E.M. + S.E.M. differencet

Tenthday 14 2-5±0-2 1-1+0-1 P<0-001Eleventhday 13 18+02 0-8+±01 P<0-001Tenth/eleventh day differencet - P< 001 P< 0 05

* Assessed on a scale of 0 (negligible activity) to 3 (very active) by 3 independent observers, whosescores were averaged for each membrane.

t By Student's t-test.

Table 4. Dimensions and surface areas of intact parietal yolk sacs at differentstages ofgestation

Age of embryo Ninth day Tenth day Eleventh day

Number measured 119 252 45Mean minimum diameter (mm± S.E.M.) 1-30+ 003 2-71 + 002 3 99 + 004Mean maximum diameter (mm± S.E.M.) 149 + 002 288 + 002 467 + 006Mean surface area (mm2 ± S.E.M.) 63 +0-2 248 + 04 587 ± 12

cell activity was seen in many preparations, this occurred without any significant netmovement of the cells; rather the cells appeared to change shape rapidly and to moveover and between each other without straying far from their original neighbours.A frequent occurrence in the cultured parietal endoderms was the appearance, on

the surface of the monolayer, of cell clumps which grew larger during the course ofthe cultures (Fig. 8).

Parietal yolk sac dimensionsMeasurements of the parietal yolk sacs used in the smooth/blebby cell analysis,

in their intact state, are given in Tables 1 and 2. However, because the number ofembryos used in this part of the study was relatively small, and rapid expansion wasan important feature of the development of the PE/R complex in vivo, the dimen-sions of larger samples of parietal yolk sacs at different stages of gestation are shownin Table 4. These dimensions tend to be slightly greater than those given in theprevious Tables because they were obtained from conceptuses isolated in theafternoon, whereas the preparations used in the other parts of the study wereisolated in the morning. Also shown in Table 4 are the surface areas of the intactPE/R complexes, calculated using the formula, Area = 4fTab, where a is the minimumand b the maximum radius of the parietal yolk sac.

DISCUSSION

The results demonstrate that parietal endoderm cells from embryos of differentages and from different regions of the same embryo show considerable heterogeneity.The cells near the placenta maintain much the same blebby appearance throughoutthe age range studied, but as the conceptus and hence the parietal yolk sac grow, sothe distal cells become increasingly smooth and rounded, ultimately acquiring

44

Mouse parietal endodermnumerous short filopodia and in some cases becoming very large. The blebby cellsnear the placenta appear to be derived in the younger embryos from large roundedcells in the extreme proximal region, which have the same morphology observedpreviously in eighth day embryos (Hogan & Newman, 1984).The functional significance, if any, of the diversity in shape of PE cells is not clear.

The main function of PE cells seems to be the synthesis of Reichert's membrane,which, because it completely envelops the fetus and visceral yolk sac, is thought toact as a coarse filter allowing access to the fetus of nutrients from the mother butexcluding entry of maternal cells (Smith & Strickland, 1981; Gardner, 1983). Themorphological differences in PE cells of different ages and in different locations maytherefore reflect quantitative or qualitative changes in basement membrane syn-thesis.The reduction in the proportion of cells exhibiting the blebby phenotype after a

6 hour period in culture is probably due to a change in the shape of individual cells,as time-lapse studies have shown little net movement of cells in vitro. That such achange in distribution of blebby cells near the placenta was not observed in vivo maybe a consequence of the rapid expansion of the conceptus; as the PE cells changefrom blebby to smooth, so they may move (or be carried) away from the placenta,and this expansion may occur preferentially in the proximal region. Circumstantialevidence in support of this hypothesis is the appearance in vitro, predominantly inthe proximal zone, of clumps of PE cells on top of the layer attached to Reichert'smembrane. These may be newly formed cells that are unable to find spaces on themembrane due to its failure to expand.The time-lapse studies show that the greatest motile activity occurs in the peri-

placental region and that it declines with increasing age. Hence the greatest activityoccurs in regions containing a high proportion of cells with the blebby phenotype,suggesting that the blebbing could be a morphological manifestation of motility:in the absence of net movement, the activity seen in these cells may result from rapidformation and retraction of blebs (it is not possible to resolve such detail using time-lapse video) as well as localised locomotion, which may be restricted by contactinhibition.

Perhaps because dividing cells are seen less often than might be expected in such arapidly expanding tissue, it has been suggested that growth of the parietal endodermis not entirely endogenous and that cells are recruited from elsewhere. In particular,Hogan & Newman (1984) have suggested that the structural appearance of the cellsat the junction of the visceral and parietal yolk sacs of the eighth day mouse embryois consistent with a scheme whereby cells detach from the epithelial sheet at themargin of the visceral endoderm and migrate onto Reichert's membrane, trans-forming into parietal endoderm cells as they do so. Hogan & Tilly (1981) hadpreviously shown that, when cultured in contact with extraembryonic ectoderm, thevisceral endoderm from seventh day mouse embryos differentiates into parietalendoderm which then synthesises large amounts of laminin and type IV pro-collagen - proteins characteristic of Reichert's membrane. In addition, Gardner(1982) has demonstrated that following their injection into blastocysts, sixth orseventh day visceral endoderm cells contribute almost exclusively to the parietalendoderm of the resulting chimaeras.A further phenomenon that needs to be explained before the growth of the

parietal endoderm can be understood is the extremely high degree of cell mixingwhich occurs amongst the cells. Use of an in situ cell marker has shown that chimaeric

45

tenth and twelfth day parietal endoderms, produced by blastocyst injection eitherwith single primitive endoderm cells or with early inner cell mass daughter-cellpairs, show intimate mixing of host and donor cells. This contrasts with the co-herent patches of donor cells seen in chimaeric visceral endoderms produced by thesame technique (Gardner, 1984). Although the intense motile activity seen in vitroin the present study was not associated with significant net movement of the cells,it is possible that in vivo, with the rapid expansion of the conceptus and appositionof visceral and parietal endoderms, this movement could contribute to cell mixing.It is also conceivable that the cells which were seen not to be in close contact withReichert's membrane - i.e. the clumps of cells in the time-lapse studies and thedividing cells visualised by scanning electron microscopy - could, in vivo, be free tomigrate in the yolk cavity, again contributing to cell mixing.

SUMMARY

The morphological appearance of parietal endoderm (PE) cells from ninth tofourteenth day mouse embryos was examined by light and scanning electron micro-scopy, and their behaviour in vitro was studied using time-lapse video recordings.At all ages, the PE cells proximal to the placenta had a predominantly blebby

phenotype whilst, in all but the youngest embryos studied, the distal cells weresmooth and rounded. In the older embryos, the cells in the middle and distal regionshad numerous short filopodia and were sometimes very large and flattened.

Time-lapse studies on tenth day preparations showed intense motile activity(though little net movement) of the PE cells in the proximal region, with relativelylittle activity in more distal regions. The cell activity was generally less in eleventhday preparations, though it was still significantly greater in the proximal zone thanelsewhere. The significance of these observations for understanding the developmentof the parietal endoderm is discussed.

I wish to thank Professor R. L. Gardner for encouragement and advice throughoutthis study, Mrs J. Williamson and Miss J. Nichols for help in preparation of themanuscript, and Mr T. J. Davies, Miss J. Green and Mrs M. Bergin-Cartwright fortechnical assistance. I am also grateful to the Imperial Cancer Research Fund forfinancial support.

REFERENCES

GARDNER, R. L. (1982). Investigation of cell lineage and differentiation in the extraembryonic endodermof the mouse embryo. Journal ofEmbryology and Experimental Morphology 68, 175-198.

GARDNER, R. L. (1983). Origin and differentiation of extraembryonic tissues in the mouse. InternationalReview ofExperimentalPathology 24, 63-133.

GARDNER, R. L. (1984). An in situ cell marker for clonal analysis of development of the extraembryonicendoderm in the mouse. Journal ofEmbryology and Experimental Morphology 80, 251-288.

HOGAN, B. L. M. (1980). High molecular weight extracellular proteins synthesised by endoderm cellsderived from mouse teratocarcinoma cells and normal extraembryonic membranes. DevelopmentalBiology 76, 275-285.

HOGAN, B. L. M., COOPER, A. & KURKINEN, M. (1980). Incorporation into Reichert's membrane oflaminin-like extracellular proteins synthesised by parietal endoderm cells of the mouse embryo.DevelopmentalBiology 80, 289-300.

HOGAN, B. L. M. & NEWMAN, R. (1984). A scanning electron microscope study of the extraembryonicendoderm of the 8th-day mouse embryo. Differentiation 26, 138-143.

HOGAN, B. L. & TILLY, R. (1981). Cell interactions and endoderm differentiation in cultured mouseembryos. Journal ofEmbryology and Experimental Morphology 62, 379-394.

46 D. L. COCKROFT

Mouse parietal endoderm 47JOLLIE, W. P. (1968). Changes in the fine structure of the parietal yolk sac of the rat placenta with increas-

ing gestational age. American Journal ofAnatomy 122, 513-522.MINOR, R. R., HOCH, P. S., KOSZALKA, T. R., BRENT, R. L. & KEFALIDES, N. A. (1976a). Organ cultures

of the embryonic rat parietal yolk sac. I. Morphologic and autoradiographic studies of the depositionof the collagen and noncollagen glycoprotein components of basement membrane. DevelopmentalBiology 48,344-364.

MINOR, R. R., STRAUSE, E. L., KOSZALKA, T. R.,BRENT, R. L. &KEFALIDES, N. A. (1976b). Organ culturesof the embryonic rat parietal yolk sac. Il. Synthesis, accumulation and turnover of collagen andnoncollagln basement membrane glycoproteins. DevelopmentalBiology 48, 365-376.

SMITH, K. K. & STRICKLAND, S. (1981). Structural componentsand characteristics ofReichert's membrane,an extra-embryonic basement membrane. Journal ofBiological Chemistry 256, 4654-4661.

WHITTINGHAM, D. G. & WALES, R. G. (1969). Storage of two-cell mouse embryos in vitro. AustralianJournal ofBiological Science 22, 1065-1068.

WISLOCKI, G. B. & PADYKULA, H. A. (1953). Reichert's membrane and the yolk sac of the rat investigatedby histochemical means. American Journal ofAnatomy 92,117-151.