PATTERNS & PHENOTYPES

Systematic Localization of Oct-3/4 to theGastrulating Mouse Conceptus SuggestsManifold Roles in Mammalian DevelopmentKaren M. Downs

Oct-3/4 was localized to the mouse conceptus between the onset of gastrulation and 16-somite pairs (-s;�6.5–9.25 days postcoitum, dpc). Results revealed Oct-3/4 in a continuum of morphologically distinctepiblast-derived embryonic and extraembryonic tissues. In the allantois, distal-to-proximal diminution inthe Oct-3/4 domain over time and co-localization with Flk-1 in angioblasts accorded with a role in vasculardifferentiation and the presence of a stem cell reservoir. In addition, visceral endoderm exhibited adynamic salt-and-pepper distribution, which, combined with previous results of fate mapping and geneexpression, suggested that Oct-3/4 is involved in the genesis of definitive endoderm. By 8-s, Oct-3/4 wasglobally down regulated in all but putative primordial germ cells (PGCs) and some allantoic cell clusters.Taken together, Oct-3/4’s expression profile suggests unexpected and potentially far more versatile roles indevelopment than have been previously appreciated. Developmental Dynamics 237:464–475, 2008.© 2008 Wiley-Liss, Inc.

Key words: allantois; endoderm; Flk-1; gastrulation; heart; hindgut; mouse; node; Oct-3/4; primordial germ cells; PGCs;pluripotency; Pou5f1; stem cells; umbilical cord; vasculogenesis

Accepted 23 November 2007

INTRODUCTION

During embryonic development, cellfate is regulated by transcription fac-tors that act as molecular controls toactivate or repress specific batteries ofgene expression. Oct-3/4, encoded byPou5f1, is thought to be one of theearliest transcription factors involvedin differentiation in the mammalianconceptus (reviewed in Stefanovic andPuceat, 2007).

Oct-3/4 is associated with toti- andpluripotent cells during early pre-im-plantation development in both miceand humans. In these species, Oct-3/4was found as maternal and embryonicprotein in the unfertilized egg, zygote,

and the zygote’s descendents throughthe blastocyst stage (Palmieri et al.,1994; Cauffman et al., 2006). Once theblastocyst formed, Oct-3/4 was down-regulated in trophectoderm, thereaf-ter being found exclusively in theblastocyst’s inner cell mass (ICM)(Palmieri et al., 1994; Cauffman et al.,2006).

Manipulation of murine embryonicstem (ES) cells provided compellingevidence that Oct-3/4 may control, in adose-dependent manner, cell fate asthe ICM segregates into epiblast andprimitive endoderm (Palmieri et al.,1994; Niwa et al., 2000). Surprisingly,for so important a gene product in po-

tency and differentiation, the compre-hensive whereabouts of Oct-3/4 inICM derivatives beyond implantationare not known.

Gastrulation begins at about 6.5dpc, and is the time when the primi-tive streak, or embryonic antero-pos-terior (A-P) axis, is established andthe three primary germ layers, ecto-derm, mesoderm, and endoderm, sys-tematically segregate from the epi-blast. Over a 48-hr period, spatialcoordinates established by the streakdirect differentiation of the germ lay-ers, and together these componentslay down the basic body plan of thefetus (Beddington, 1983).

Department of Anatomy, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WisconsinGrant sponsor: National Institutes of Health; Grant number: RO1HD042706.*Correspondence to: Karen M. Downs, Department of Anatomy, University of Wisconsin-Madison School of Medicine andPublic Health, 1300 University Avenue, Madison, WI 53706. E-mail: kdowns@wisc.edu

DOI 10.1002/dvdy.21438Published online 16 January 2008 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 237:464–475, 2008

© 2008 Wiley-Liss, Inc.

In light of the importance of gastru-lation and Oct-3/4’s role in regulatingdevelopmental potency, I have inves-tigated the whereabouts of Oct-3/4protein in the mouse conceptus. Pre-vious localization to the gastrula be-tween 6.0 and 11.0 dpc painted broadbrushstrokes of Oct-3/4 mRNA, de-scribing it in epiblast, neurectoderm,and the base of the allantois, whereexpression was reputed to be in pri-mordial germ cells (PGCs) (Scholer etal., 1990). Findings here reveal thatthe number of Oct-3/4-expressingsites during gastrulation far exceededthose previously reported. As the pri-mary germ layers were deployed fromthe epiblast, they, and subsequentlytheir own progeny, exhibited robustlevels of Oct-3/4. Over time, Oct-3/4diminished, leaving just two clear pos-itive cell populations, putative PGCs,and hitherto unclassified allantoiccells.

These results provide a comprehen-sive and fundamental spatiotemporalblueprint for evaluating the signifi-cance of Oct-3/4’s gene activity in dif-ferentiating cells and tissues. Fur-ther, they point to tissues that may,on the basis of Oct-3/4 expression, beused to create unique stem cell lines.

RESULTS

Immunostaining WholeMount Prepared SpecimensProduced Superior Oct-3/4Signal

Several previous studies from my lab-oratory revealed variability in the re-sults of immunostaining according tothe type of fixative used. For example,AHNAK and T signals in sectionsof Bouin’s-fixed specimens exceededthose obtained in paraformaldehyde(Downs et al., 2002; Inman andDowns, 2006a). Thus, in this study, Icompared the robustness of Oct-3/4 inBouin’s- or paraformaldehyde-fixedand sectioned material with that inwhole mounted specimens fixed in 4%paraformaldehyde/methanol followedby immunostaining and sectioning.

Immunostaining sections of mate-rial fixed in paraformaldehyde yieldedno Oct-3/4 signal (see ExperimentalProcedures section; data not shown).

By contrast, Bouin’s-fixed specimensexhibited an Oct-3/4 signal that,whilst localized to the same sites asthose in whole mount preparations,was nevertheless inferior in intensityand more diffuse than that in wholemount preparations (compare Fig.1A,B). Subsequently, for each stageexamined here, all material was im-munostained in whole mounted speci-mens prior to embedding and sec-tional analysis.

Pre-binding the Oct-3/4 antibodywith control peptide for 10 hr at 4°Cas well as immunostaining in the ab-sence of antibody to Oct-3/4 revealedspecificity for all anatomical sites ex-cept trophoblast giant cells and pari-etal endoderm (Fig. 1C–E, and datanot shown). In all sites identified here,and at all stages, Oct-3/4 localizedsolely to nuclei.

Early Gastrula: StreakStages (�6.5–7.0 dpc)

At the onset of gastrulation (Early-and Mid-Streak, ES, MS stages;�6.5–6.75 dpc), Oct-3/4 positive tis-sues were the epiblast, primitivestreak, embryonic and extraembry-onic mesoderm, and embryonic vis-ceral endoderm (EVE) (Fig. 2A–C).The Oct-3/4 EVE domain extended cir-cumferentially around the egg cylin-der, from the anterior level of theprimitive streak to the distal tip of theegg cylinder (Fig. 2A–C).

At the Late Streak (LS) stage (�7.0dpc), the only new Oct-3/4-positivestructure was the node as it emergedat the anterior end of the primitivestreak (Fig. 2D). Oct-3/4-positive EVEcells were now associated with the fullcircumference of paraxial and lateralplate mesoderm. With the exception ofextraembryonic mesoderm, all ex-traembryonic tissues were negativeduring the streak stages.

No Bud Stage(�7.0–7.25 dpc)

At the No Bud (OB, No Allantoic Bud)stage (�7.0–7.25 dpc), the notochordwas elongating through the midlinefrom its origin in the node, and con-tained intensely positive Oct-3/4 cells(Fig. 2E). On either side of this struc-ture, EVE cells were also positive,

overlying prospective paraxial andlateral plate mesoderm (not shown forthis stage, but see the LB stage, Fig.2I,J, below). Posterior lateral platemesoderm and nascent prospectiveparaxial mesoderm themselves wereintensely positive (compare Fig.2E,F). By contrast, anterior-half lat-eral plate mesoderm was of mixed in-tensity, varying from slightly positiveto negative (Fig. 2E).

In the extraembryonic region, ex-traembryonic mesoderm lining thenascent exocoelom contained Oct-3/4(Fig. 2G), as did amniotic ectoderm(Fig. 2G). All other extraembryonictissues were negative.

Neural Plate/Allantoic BudStages (�7.25–7.5 dpc)

At both the early (EB, �7.25 dpc) andlate allantoic bud (LB, ��7.5 dpc)stages, Oct-3/4 patterns were similar(Figs. 2H–J, 3A,B). In the embryonicregion, Oct-3/4 was still robust in theepiblast, primitive streak, and node(Fig. 2H). The notochord had reachedits full length, but anterior cells wereeither negative, or less intensely pos-itive than posterior ones (Fig. 2H).

Paraxial mesoderm was darklystained (Fig. 2I), but only posteriorlateral plate mesoderm exhibited ho-mogeneous Oct-3/4 (Fig. 2J). Oct-3/4-positive EVE was associated withparaxial and lateral plate mesoderm(Fig. 2I,J) and, in a minority of speci-mens (EB stage, 20%, N � 5; LB stage,40%, N � 5; not shown), began to ex-tend to posterior primitive streak-as-sociated EVE. The neural plate con-tained Oct-3/4 (Fig. 2I).

In the extraembryonic region, exo-coelomic mesoderm was still positive(Fig. 3A,B), but nascent yolk sac bloodislands did not exhibit Oct-3/4 at this,or at any other, time (not shown). Theallantoic bud had appeared and, dur-ing the bud stages, its base was bothcontinuous with the underlying prim-itive streak and adherent to overlyingextraembryonic visceral endoderm.Henceforth, allantois-associated ex-traembryonic visceral endoderm shallbe referred to as “AX.” Bud-stage al-lantoises were examined in sagitalorientation, the base of the allantoistaken as its site of insertion into thevisceral yolk sac and amnion, as pre-viously described (Downs and Har-

OCT-3/4’S EXPRESSION PROFILE IN MOUSE 465

Fig. 1. Comparison of Oct-3/4 immunohistochemistry; the posteriorprimitive streak/allantois boundary in transverse orientation. In this andall other panels, histological sections were lightly counterstained withhematoxylin (purple color); Oct-3/4 protein is indicated by brown color.In A, both sets of orthogonal arrows indicate that, unless otherwisenoted, anterior (a) is on the left, and posterior (p) is on the right in allpanels in this study. Lower left orthogonal arrows provide distal (d)/proximal (p’) coordinates for the embryonic portion of the egg cylinder,located below the amnion (am); upper right orthogonal arrows providesimilar distal/proximal coordinates for the extraembryonic portion abovethe amnion. In whole mount specimens, the anterior yolk sac wasopened prior to immunostaining. A–E: Sagital orientation. F–G: Trans-verse orientation. I: Frontal orientation. A: EHF, Bouin’s fixed, sectioned,and immunostained with anti-Oct-3/4. B–I: Whole mount immunostainedpreparations followed by sectioning. B–E: From the same control exper-iment. B: EHF, 4% paraformaldehyde/methanol whole mount immuno-histochemistry with anti-Oct-3/4. C: EHF, anti-Oct-3/4 was placed at4°C for 10 hr prior to use. D: EHF, anti-Oct-3/4 � control Oct-3/4peptide, incubated at 4°C for 10 hr prior to use. E: 2-s, minus anti-Oct-3/4. Controls reveal that the brown stain in trophoblast giant cells (gc) isnon-specific. F–H: 2-s, three transverse sections illustrating the poste-rior primitive streak (pps) (F); the boundary between the pps and allantois(pps/al) (G); and the first section in the base of the allantois (al) (H). I: 2-s,frontal orientation. The three long horizontal lines indicate the three 6-�mlevels at which the sections in F–H were taken, with the shortest line atthe top corresponding to the base of the allantois in H, the middle line tothe al/pps in G, and the lower line to the pps in F, the latter two of whichare of similar length (see text for details). Scale bar in E � 200 �m (A–E);in H � 50 �m (F–H), in I � 50 �m (I). � Ab, presence of anti-Oct-3/4antibody; �Ab, absence of anti-Oct-3/4 antibody; al, allantois; am,amnion; ax, allantois-associated visceral endoderm; cp, control peptide;eve, embryonic visceral endoderm; gc, trophoblast giant cells; hf, head-folds; pps, posterior primitive streak.

Fig. 2. Oct-3/4 in streak and neural plate stages. A: MS, sagital sec-tion. Thin horizontal line through the posterior epiblast and overlying EVEindicates the anteriormost limit of the primitive streak at this stage.Anterior and posterior arrowheads indicate the extent of Oct-3/4 cells inEVE. B,C: MS, transverse sections 54 (B) and 30 (C) �m, respectively,from the distal embryonic tip of a second conceptus. White asterisk in B,primitive streak; in C, the streak is not present. Arrowheads indicateexamples of distinct Oct-3/4-positive cells within the EVE. D: LS, sagitalsection. Arrowheads as in A. The streak has now reached its full length,and its anterior end has condensed into the node (arrow). E: OB, sagitalsection of anterior half and distal tip, including the node (arrow). Anteriorarrowhead indicates the anteriormost limit of Oct-3/4 cells in the EVE/notochord. Note that the intensity of Oct-3/4-positive EVE cells along itsanterior length is similar to that of the epiblast. Also note that lateral platemesoderm (lpm) between the EVE and epiblast is mottled, exhibitingpositive and negative Oct-3/4 cells, whilst paraxial mesoderm (pm) ishomogeneously positive. F: OB, sagital section of posterior region,arrowhead indicates the posteriormost extent of Oct-3/4 within EVE,which overlies the full circumference of lpm. G: OB, nascent exocoelo-mic cavity (x) lined with Oct-3/4-positive extraembryonic mesoderm. H:EB, sagital section through anterior half and distal tip, including the node(arrow). Arrowhead indicates the anteriormost extent of Oct-3/4-positivecells, whose intensity was diminishing with respect to more posteriornotochordal cells and the strongly positive node and epiblast. I: LB,transverse section through paraxial mesoderm, and including the node(arrow) and overlying neural plate (np). Posterior is on the bottom,indicated by the primitive streak (asterisk). Arrows indicate that Oct-3/4-positive cells lie in the overlying EVE’s full circumference. J: LB,transverse section through lpm of the same conceptus as I, including theprimitive streak (asterisk). Some notochordal cells (nt), located oppositethe streak in this section, still exhibited Oct-3/4. Arrowheads as in I.Scale bar in J � 50 �m (A, D–I); 75 �m (J); in C � 50 �m (B, C). e,epiblast; lpm, lateral plate mesoderm; np, neural plate; nt, notochord.pm, paraxial mesoderm; x, exocoelomic cavity; xm, extraembryonicmesoderm.

466 DOWNS

mann, 1997) (Fig. 3A,B; also see Ex-perimental Procedures section). Bothinner and outer allantoic cell popula-tions contained Oct-3/4 along the en-tire allantoic length (25–87 �m) (Figs.3A,B, 4A,B). The AX itself was nega-tive for Oct-3/4 at this time (Fig. 4C).All other extraembryonic cells werenegative (data not shown).

Headfold Stages(�7.75–8.0 dpc)

At the early headfold (EHF) stage(�7.75 dpc), the newly formed ante-rior neurectoderm and surface ecto-derm exhibited Oct-3/4 (Fig. 1B,C). Inthe node, Oct-3/4 localized to all butits ventral anterior aspect, where Oct

3/4 was now undetectable (Fig. 5A,B).The EVE pattern of Oct-3/4 had alsoshifted, and was now confined to theposterior half of the embryo, includingthe posterior primitive streak. Allother posterior embryonic cells andtissues were positive.

In the extraembryonic region, Oct-3/4 was found predominantly in inner,and occasionally outer, associatedcells of the allantois (Fig. 3C). Allan-toises were now analyzed for theircontent of Oct-3/4 in transverse orien-tation (for details, see ExperimentalProcedures section); by the EHFstage, most (83.3%) were consistently

Fig. 3.

Fig. 3. Oct-3/4 in the allantois and allantoicvasculature, associated visceral endoderm, andhindgut endoderm. A: EB, sagital profile. B: LB,sagital profile. Oblique lines in A,B connect thesite of insertion of the allantois into the amnionand yolk sac, and define the base of the allan-tois as previously described (Downs and Har-mann, 1997), and the proximal level from whichthe base to the distal tip was measured forFigure 4. C: EHF, sagital profile. Arrowheadsindicate Oct-3/4-positive cells in allantois-asso-ciated visceral endoderm (ax) (top arrowhead)and EVE (eve) overlying the posterior primitivestreak (bottom arrowhead). The horizontal lineindicates the level above which the length of theOct-3/4 domain was measured in transversesections from this stage onward; note that itincludes those Oct-3/4 cells adjacent to the AX.For further details, see text and ExperimentalProcedures section. D: 1-s, transverse section.Oct-3/4 positive amniotic ectoderm (arrows onam) and AX (arrowheads) are particularly clearin this section. E: 5-s, semi-oblique sagital/fron-tal section. The thin line extends across theboundary between the base of the allantois andthe embryonic region, as previously defined inA,B, above; thus, the Oct-3/4-positive portionof the allantois and Oct-3/4-positive hindgut arephysically continuous. Arrowheads indicateOct-3/4-positive cells in AX (top arrowhead)and in EVE overlying the embryonic hindgut(bottom arrowhead). F: 5-s, sagital section re-vealing the association between Oct-3/4-posi-tive allantois, AX cells, and the primary umbilicalvessel (asterisks). G–J: 2-s. Consecutive seriesof transverse sections at specific levels throughan X-gal-stained Flk/LacZ allantois, the angio-blasts of which are royal blue (purplish color ishematoxylin), immunostained with anti-Oct-3/4(brown). In all panels, arrows indicate Flk/Oct-3/4-positive cells; arrowheads in G indicateOct-3/4-positive cells in the AX; 18 (G), 60 (H),and 72 (I) �m from the boundary with the pos-terior primitive streak. Insets (I) are examples ofX-gal-positive cells from this section either co-stained with Oct-3/4 (top inset), or negative forOct-3/4 (bottom inset). J: 150 �m from thestreak boundary. Scale bar in F � 50 �m (A–F),in J � 50 �m (G–J). hg, hindgut; ps, primitivestreak; xve, extraembryonic visceral endoderm.

OCT-3/4’S EXPRESSION PROFILE IN MOUSE 467

directed toward the chorion. By theLHF stage, all allantoises were consis-tently pitched toward the chorion.

At the EHF stage, the length of theallantois had exceeded 100 �m in83.3% of specimens (Fig. 4A) and, inthese, Oct’s distal domain was short-ening relative to the total allantoiclength, becoming confined to the mid-and proximal thirds (Figs. 3C, 4B). Intransverse sections, the Oct-3/4 do-main appeared symmetric about theallantois’s putative A-P axis, butasymmetric with respect to its dorsal-ventral (D-V) one (data not shown forthe headfold stages, but see 1-s stage,Fig. 3D). The more ventral portion ofthe allantoic Oct-3/4 domain wasclosely apposed to the overlying AX,where Oct-3/4 was observed for thefirst time at the EHF stage (Figs. 3C,4C).

In the remaining 16.7% of EHF-stage material (N � 1, not included inFig. 4 calculations; see ExperimentalProcedures section), the length of theallantois was 80 �m, similar to thatfound at the LB stage (Fig. 4A). In thisspecimen, the Oct-3/4 domain occu-pied the entire length of the allantois,

similar to the earlier EB and LBstages. Thus, 87–100 �m must be thecritical allantoic length at which re-duction in the Oct-3/4 domain occurs.By contrast with the previous stages,however (Fig. 4B), the AX in thisEHF-stage specimen exhibited sixOct-3/4-positive cells, suggesting thatemergence of AX Oct-3/4 cells isstage-, rather than length-, depen-dent.

Early Somite Stages(1–7-Somite Pairs, -s;�8.0–8.5 dpc)

The posterior aspect of the node re-mained positive through 5-s (Fig.5C,D). A series of transversely ori-ented specimens highlighted the inti-mate association between the poste-rior ventral portion of the node, andEVE (Fig. 5E–H), both of which con-tained Oct-3/4 (Fig. 5D–G). Further-more, Oct-3/4-positive EVE was con-fined to that overlying the embryonicprimitive streak.

Anterior and posterior neurecto-derm exhibited robust levels of Oct-3/4 until 5-s (Fig. 6A). By 6-s, levels of

Fig. 5. Oct-3/4 in the node and associated EVE. A: EHF, sagital section through the node. Arrowpoints to the Oct-3/4-negative anterior ventral region, and the arrowhead points to the Oct-3/4-positive crown (Bellomo et al., 1996) of the node. B: EHF, frontal section of another conceptusthrough the Oct-3/4-positive posterior crown (arrowhead). C: 1-s, sagital section. Arrow andarrowhead as in A and B. D: 5-s, sagital section through the posterior half of the embryonic region.The paired asterisks/arrowheads indicate the extent to which the posterior node was intimatelyassociated with adjacent EVE, better seen in the series in E–H. Arrows as in A and C. E–H: 4-s,serial transverse sections through the node, each panel separated by 18–36 �m. E: Anterior ventralnode (arrow) was negative for Oct-3/4, whilst the dorsal node (asterisk here and in all subsequentpanels) was positive. The adjacent EVE was negative at this location. F: This region of the node(arrowhead) represents the boundary between the anterior and posterior segments, and containsmixed Oct-3/4-negative and -positive cells; the adjacent EVE was Oct-3/4-positive. G: The pos-terior ventral node (arrowhead) and posterior dorsal node (asterisk). Adjacent EVE was deeplypositive. H: The node has disappeared, and the putative primitive streak is now physically contin-uous with overlying Oct-3/4-positive EVE (thin arrow). Scale bar in H � 50 �m (E–H), 39 �m (A, C),28 �m (B, D).

Fig. 6. Diminution of Oct-3/4 in embryonic and extraembryonic tissues. A: 5-s, sagital profileillustrates that, with the exception of neurectoderm and the amnion, epiblast-derived anteriortissues do not exhibit Oct-3/4 whilst posterior embryonic structures, the allantois and amnion, arehighly positive. Arrowhead points to Oct-3/4-positive cells in AX overlying the primary umbilicalvessel (asterisk). B: 5.-s, sagital section through somites. The most recently formed posteriorsomite (right arrow) contains more Oct-3/4 than an older anterior one (left arrow). C: 6-s, transversesection showing reduced Oct-3/4 in anterior neurectoderm (an) by comparison with posteriorneurectoderm (pn). D: 7-s, transverse section showing further diminution in Oct-3/4 in anteriorneurectoderm. Note the location of putative PGCs (arrowhead) in the ventral aspect of the hindgut.E: 8-s, frontal section showing the connection between the allantois and posterior end of theembryo. Putative PGCs (arrowheads), ventral aspect of the hindgut. F,G: 10-s, oblique sagitalsections showing Oct-3/4-positive cells (arrowheads) in the hindgut (hg), surface ectoderm (se),and the allantois (F), and clusters of Oct-3/4-positive cells (arrowhead) in the allantois (G). H: 16-s,sagital section through the allantois shows a cluster of Oct-3/4-positive cells (arrowhead). Scale barin H � 50 �m (B, H); 86 �m (C, D); 100 �m (E, G); 200 �m (F). Scale bar in A � 200 �m (A). bi, bloodislands; fg, foregut; ht, heart; ne, neurectoderm; s, somite; se, surface ectoderm.

Fig. 4. Oct-3/4 in the allantois and AX. In allpanels, error bars are the standard error of themean. A: Average length of the allantois and ofthe proximal allantoic region occupied by Oct-3/4, in micrometers (�m), as a function of stage.The numbers associated with each stage arethe number of specimens examined in wildtype(“F2,” top number) and Flk1/LacZ (bottom num-ber) specimens for all three panels. The range ofallantoic lengths for each stage, wildtype andFlk1/LacZ combined, were: EB: 25–40 �m; LB:40–87 �m; EHF: 132–216 �m; LHF: 240–270�m; 1-s: 198–240 �m; 2-s: 246–384 �m; 3-s:282–378 �m; 4-s: 282–462 �m; 5-s: 330–504�m; 6-s: 456–468 �m; 7-s: 510–516 �m. B:Average percent of the total length of the allan-tois occupied by proximal Oct-3/4 by stage.Where N � 3 at the EHF and 4-s stages, thedomain occupied by Oct in Flk1/LacZ allan-toises was statistically equivalent to that in wild-type (see text). C: Average number of Oct-3/4-positive cells in AX as a function of stage; F2wildtype and Flk1/LacZ were combined.

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Oct-3/4 diminished, with anteriorneurectoderm losing this protein inadvance of posterior neurectoderm(Fig. 6C). Precocious loss of anteriorOct-3/4 was particularly striking at7-s (Fig. 6D). As somites emerged fromparaxial mesoderm, they were ini-tially slightly positive, but, with age,their intensity lessened with rostral-to-caudal directionality (Fig. 6A,B).Nascent hindgut endoderm, locatedbeneath the allantois, contained Oct-3/4 (Fig. 3E).

In the extraembryonic region, amni-otic ectoderm was positive through 5-s(Fig. 3A–F) whist the mesodermal lin-ing of the exocoleomic cavity graduallybecame Oct-3/4-negative. The proxi-mal allantoic Oct-3/4 domain was as-sociated with the AX (Fig. 3C), hind-gut (Fig. 3E), and the umbilicalvasculature (Fig. 3E,F). Until 6-s, al-lantoic contact with the AX persisted(Fig. 3C–F, and not shown), with theAX’s Oct-3/4-positive cell contenthighest at 2- and 4-s (Fig. 4C).

Through 3-s, the absolute length ofthe Oct-3/4 domain increased (Fig.4A). However, despite increasing al-lantoic length (Fig. 4A), the propor-tional length of the Oct-3/4 domain inthe mid- and proximal allantoic re-gions was fairly constant through 3-s,after which expression diminishedwith increasing embryonic age, be-coming confined to the proximal quar-ter of the allantois’s full length by 6-s(Fig. 4B). Persistence of the proximalOct-3/4 allantoic domain suggestedthe presence of a stem cell reservoir, anotion supported by previous resultsthat revealed pluripotent cells in thisregion (Downs and Harmann, 1997).Moreover, distal-to-proximal diminu-

Fig. 5.

Fig. 6.

tion in the Oct-3/4 domain relative tothe increased length of the allantoiswas reminiscent of allantoic vascular-ization, which occurs with distal-to-proximal polarity throughout this pe-riod (Downs et al., 1998). Lastly, Oct-3/4 was associated with the allantoicvasculature (Fig. 3E,F). Together,these observations suggest that Oct-3/4 is involved in allantoic vasculogen-esis.

To discover Oct’s potential role inallantoic vascularization, Flk-1-con-taining allantoic angioblasts (Downset al., 1998) were co-stained for Oct-3/4 in Flk-promoter-driven LacZtransgenic conceptuses. At EHF and4-s stages (N � 3), the average lengthof the allantois and Oct-3/4 domainfell within those reported for wildtype(Fig. 4A, B; P � 0.56, EHF; P � 0.44,4-s stage; two-way Student’s t-test,equal variances assumed), demon-strating that the presence of the lacZtransgene had no effect on allantoicgrowth. Moreover, the average lengthof the Oct-3/4 domain was similar inboth genetic backgrounds (EHF: P �0.28; 4-s: P � 0.22). During the entireperiod examined (EHF-5-s stages),doubly positive Oct-3/4/Flk-1 cellswere identified (e.g., Fig. 3G–J). AllOct-3/4 cells bearing Flk-1 weremildly, rather than intensely, brown(Fig. 3I, insets).

Late Somite Stages(8–16-s; �8.75–9.25 dpc)

By 8-s, Oct-3/4 was found in just a fewcell types, including the putative germcell population, some clusters of hith-erto unidentified cells throughout theallantois, and an occasional surface ec-todermal cell (Fig. 6E). Within the hind-gut, Oct-3/4-positive cells were localizedto the ventral, rather than dorsal, re-gion (Fig. 6D,E). Specimens examinedat 10-, 12-, 14-, and 16-s pairs exhibitedsimilar patterns and intensity of stain-ing, with no new positive cell typesemerging (Fig. 6F–H). Within the allan-tois, Oct-3/4-positive cells were roundand generally clustered.

DISCUSSION

Summary of Oct-3/4 in theMouse Gastrula

Results here demonstrate Oct-3/4’spresence as an uninterrupted contin-

uum in mouse epiblast and its prog-eny tissue as the latter emerge andmature within the gastrula. Epiblast,the primitive streak, node, notochord,nascent embryonic mesoderm, neuralplate, mid- and distal EVE, nascentextraembryonic mesoderm, and amni-otic ectoderm exhibited intenselystained Oct-3/4 cells during pre-allan-toic bud stages. By bud stages, newpositive structures, neurectoderm,surface ectoderm, proximal posteriorEVE, and the allantois emerged. Oct-3/4 diminished in the notochord andanterior lateral plate mesoderm. Atheadfold stages, only the AX emergedas a new Oct-3/4-positive tissue. Otherswere losing Oct-3/4, including the ante-rior node, anterior EVE, and distal al-lantois. During 1–7-s stages, the onlynew positive structure was the hindgut;Oct-3/4 was down-regulated in paraxialmesoderm-derived somites, the walls ofthe exocoelom, and the distal-to-mid-al-lantoic regions. In specific structures,Oct-3/4 became confined to the poste-rior, or caudal node, that EVE overlyingthe caudal node and embryonic primi-tive streak, the AX, and the core of theproximal allantois. Between 8–16-sstages, Oct-3/4 was almost globally ex-tinguished, being detectable only in pu-tative PGCs of the hindgut, an occa-sional surface ectodermal cell, andsmall cell clusters in the allantois.

Oct-3/4 was limited to nuclei. Dur-ing ES, MS, and LS stages, these wereuniformly dark in positive tissues. Be-tween the OB-8-s stages, Oct-3/4’s in-tensity ranged from almost black tolight brown in most tissues. However,for reasons that are not clear, amongstpositive tissues, only the epiblast,primitive streak, neurectoderm, pos-terior embryonic mesoderm, and am-niotic ectoderm seemed uniformlypositive throughout their periods ofexpression. Two tissue subdomains,the core domain within the base of theallantois and the caudal node, alsoseemed homogeneously positive at theoutset of their expression. At �8-s,staining was either intensely dark orundetectable.

Although the nature of later-stageallantoic Oct-3/4 cells is not known,their shape and clustering were remi-niscent of yolk sac blood islands (Haarand Ackerman, 1971). Patency of theallantoic vasculature with the yolk sacand fetal arterial systems begins at

the 4-s stage (Downs et al., 1998), yetit is unlikely that extrinsic hematopoi-etic cells entered the allantois via thevasculature. Not only was Oct-3/4 un-detectable in yolk sac blood islands/circulating blood cells, but allantoicclusters were not contained withinblood vessels. Rather, allantoic Oct-3/4 cells may represent an intrinsicand, possibly, emerging definitive he-matopoietic population (Zeigler et al.,2006).

Previous results of fate mapping sug-gest that gradual extinction of Oct-3/4corresponds with increasing cell ageand differentiation. This was best illus-trated in the notochord/foregut (Fig. 2E,H) (Beddington, 1994; Tam et al., 2004),paraxial mesoderm/somites (Fig. 6A,B)(Tam and Beddington, 1987), lateralplate mesoderm/heart (Figs. 2A,J, 6A)(Kinder et al., 1999), and the allantois/umbilical vasculature (Fig. 3A–C,E,G–J) (Downs and Harmann, 1997;Kinder et al., 1999). For reasons thatare not yet understood, near-global ex-tinction of Oct-3/4 beyond 7-s (Fig. 6E)demonstrates that Oct-3/4 is not re-quired for growth and morphogenesis ofindividual organ systems (Kaufman,1992).

Oct-3/4 Plays a Major Rolein Building the UmbilicalVasculature

Oct-3/4 triggers expression of meso-dermal and cardiac-specific genesthrough Smad2/4 in embryonic stem(ES) cells, driving the latter towarddifferentiation into cardiomyocytes(Zeineddine et al., 2006). In accordwith this conclusion in vitro, Oct-3/4was highly expressed in prospectiveheart mesoderm, which emerges fromthe primitive streak at the MS stage(Kinder et al., 1999). Thus, results ofthis study support a role in vivo forOct-3/4 in heart development.

In addition, results revealed thatOct-3/4 is involved in vasculariza-tion of the allantois. The allantois,which is the precursor tissue of theumbilical cord, undergoes de novovasculogenesis (Downs et al., 1998;Drake and Fleming, 2000). Flk-1-positive angioblasts were initiallymost abundant in the distal allantoisat the LB/EHF stages (Downs et al.,1998), where allantoic cells are old-

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est (Downs and Harmann, 1997;Kinder et al., 1999).With distal-to-proximal directionality, Flk-1-posi-tive angioblasts emerged down thelength of the allantois such that, by4 – 6-s, a primary Flk-1-positive um-bilical vasculature was formed(Downs et al., 1998, 2004; Inmanand Downs, 2006b, 2007). These ob-servations led to a model of polarizedallantoic vascularization (Downs etal., 2004). Intriguingly, as Flk-1 isacquired with distal-to-proximal ki-netics (Downs et al., 1998), Oct-3/4diminished with the same direction-ality (Fig. 4A,B). This correlationlasted throughout vasculogenesis,with many non-robustly-stained Oct-3/4 cells exhibiting Flk-1. Moreover,persistence of Oct-3/4 within theproximal core domain strongly ar-gues for this region as a stem cellreservoir for building the differenti-ating allantoic vasculature. This re-gion contains Brachyury, or T (In-man and Downs, 2006a), which, asrecently demonstrated, is requiredfor allantoic vascularization and pat-terning (Inman and Downs, 2006b)On the basis of these findings, I con-clude that Oct-3/4 plays a significantrole in formation of the umbilicalvasculature.

Contribution of Oct-3/4-Positive Cells to GutEndoderm

The origin of definitive gut endodermis complex, but involves intercalationof streak- (Lawson et al., 1986, 1991;Lawson and Pedersen, 1987) and epi-blast- (Tam and Beddington, 1992) de-rived definitive endoderm into theEVE. As a result, gene expression inthis cell layer might be expected, incertain cases, to exhibit salt-and-pep-per distribution. Oct-3/4-positive cellswere located amongst unstained EVEcells through 6-s (Fig. 7). Recently, thechick homolog, PouV/Oct-3/4, exhib-ited similar expression patterns in thehypoblast, the chick equivalent ofmouse visceral endoderm (Lavial etal., 2007). Results of fate mapping,gene expression, and ablation studies(reviewed in Lewis and Tam, 2006)demonstrated that genesis of defini-tive gut endoderm involves a varietyof gene activities and morphogeneticmovements, as descendents of thebud-stage node and three EVE re-gions, “posterior-distal,” “posterior-middle,” and “posterior-proximal,” be-came part of the embryonic gut (Tamet al., 2004). In the present study, allof these exhibited Oct-3/4 (e.g., Figs.

2H–J, 7). Moreover, Oct-3/4’s expres-sion pattern in EVE was uncannilysimilar to that of Cerberus betweenthe LS and LB stages (Fig. 7B)(Lewis and Tam, 2006). Together,these observations support a role forOct-3/4 in development of definitiveendoderm.

The significance of Oct-3/4 in AX isless clear. Oct-3/4-positive cells wereapparent in this tissue at the EHFstage, and were present until 6-s, af-ter which this endoderm was nolonger clearly associated with the al-lantois. As the AX was found in theextraembryonic region, it may becomepart of the visceral yolk sac. On theother hand, the AX contained Oct-3/4-positive cells, distinguishing it fromall other extraembryonic visceralendoderm, and suggesting that, likethe EVE, it will become part of theembryonic hindgut. EVE’s ability toshift its position (Thomas and Bed-dington, 1996) provides a fitting para-digm for building the hindgut by AXtranslocation. Whether the AX be-comes part of the hindgut is, however,currently unknown, but loss of obvi-ous physical association between theallantois and AX by 7-s lends supportto this possibility.

Fig. 7. Summary of Oct-3/4 in visceral endoderm over time. Schematic diagram of the embryonic portion of the egg cylinder, proximal allantois, andAX. The primitive streak is indicated by a heavy black line. The mottled grey color in all of the panels represents Oct-3/4 expression in visceralendoderm (eve, ax)/notochord (nt). A: ES, MS stages, representing a period of about 12 hr, Oct-3/4 is localized to the distal half of the egg cylinder,anterior to the primitive streak. B: LS-LB stages, representing about 12 hr, Oct-3/4 is found in the distal half of the egg cylinder, the entire node (*, andheavy line), the notochord (nt), and EVE overlying the primitive streak at the level of lateral plate mesoderm (lpm) and paraxial mesoderm (pm). Thecircles over the posterior primitive streak indicate that only 20 and 40% of EB and LB stage specimens, respectively, exhibited Oct-3/4 in EVE in thisposterior region (see text for details). *’, *”, and *”’ are the “posterior-distal,” “posterior-middle,” and “posterior-proximal” regions fate-mapped by Tamet al. (2004). See text for details. C: At the headfold stages, representing about 4–6 hr, Oct-3/4-positive EVE was confined to the posterior regionoverlying the full extent of the primitive streak. In addition, Oct-3/4 localized to the AX. Half-circle, delineated by heavy line, represents the caudal node.The arrow points to the Oct-3/4-positive proximal allantoic domain, outlined by a thick line. D: By 1-s, Oct-3/4 was confined to EVE overlying theremainder of the embryonic primitive streak, and AX. Arrowhead, node; arrow as in C.

OCT-3/4’S EXPRESSION PROFILE IN MOUSE 471

The Caudal Node: A StemCell Reservoir?

By 7.75 dpc, Oct-3/4 was found inneurectoderm and surface ectoderm.In the node, Oct-3/4 was present in allbut its ventral anterior aspect. Re-sults of elegant tour-de-force potencystudies (Cambray and Wilson, 2007)have provided compelling evidencethat the anterior portion of the node isnot self-renewing; rather it contains alimited reservoir of cells that contrib-utes to the notochord. By contrast,other results have demonstrated thatthe ventral node contains a self-re-newing cell population that acts as astem cell reservoir for building mid-line embryonic structures (Bedding-ton, 1994; Tam et al., 2004). Moreover,in the chick, fate mapping revealedthat Henson’s node, analogous to thatof the mouse (Beddington, 1994), hasa complex architecture, various partsof which give rise to multiple cell lin-eages, including endoderm and neuraltube (Selleck and Stern, 1991). To-gether, these results argue that thecaudal node, conspicuously Oct-3/4-positive in this study, may be a stemcell reservoir, providing raw materialsto expand posterior neurectoderm andthe gut during later embryonic stages.

Inconsistencies BetweenOct-3/4’s Presence and“Stemness” In Vivo

Neural plate/LB anterior epiblast isfated to give rise to anterior neurecto-derm (Beddington, 1982). Moreover,anterior epiblast loses pluripotency bythis stage, failing to contribute to gutendoderm, and preferentially coloniz-ing neurectoderm (Beddington, 1982).Thus, despite robust expression ofOct-3/4 (Fig. 2H–J), anterior epiblastis developmentally restricted duringearly gastrulation. By contrast, distaland posterior epiblast were pluripo-tent (Beddington, 1982) and, in thisstudy, they also exhibited high levelsof Oct-3/4 (Fig. 2H–J).

In addition, Oct-3/4 was expressedin both the mid- and proximal allan-toic regions until 5-s. Yet, previousheterotopic grafting experiments re-vealed that the mid-allantoic region ofthe headfold-stage conceptus colo-nized only embryonic blood vessels. Bycontrast, the proximal allantoic region

contained cells that colonized deriva-tives of all three primary germ layers(Downs and Harmann, 1997). To-gether, these data suggest that thepresence of Oct-3/4 is not an accurateindicator of pluripotency.

Recently, Oct-3/4’s correlation withpluripotency was challenged in sev-eral adult settings, ranging from dif-ferentiated adult human peripheralblood mononuclear cells that con-tained Oct-3/4 (Zangrossi et al., 2007),to pluripotent adult stem cells that didnot (Ledford, 2007). Together with theaforementioned findings, these con-tradictions raise the serious questionof whether Oct-3/4’s presence/absenceaccurately and universally reflects thepluripotent/differentiated state.

In Vitro, Oct-3/4 ExpressionAccords With Oct-3/4-PositiveTissue Origin

Despite inconsistencies in vivo, Oct-3/4’s absence/presence in a variety ofstem cell lines in vitro accords with de-velopmental restriction/pluripotency andeach cell line’s in vivo precursor tissue.For example, trophoblast stem cells,or TSCs, colonize only trophectodermderivatives and do not express Oct-3/4(Tanaka et al., 1998). In vivo, Oct-3/4is down-regulated in trophectoderm atimplantation (Nichols et al., 1998;Niwa et al., 2000). Moreover, trophec-toderm-derived chorionic ectoderm, asource of TSCs during gastrulation(Uy et al., 2002), does not exhibit Oct-3/4 mRNA (Scholer et al., 1990) (Figs.1–3, 5). In addition, extraembryonicvisceral endoderm, or XEN, stem cellsare also developmentally restrictedand do not express Oct-3/4 (Kunath etal., 2005). With the exception of theAX (Fig. 3C–G), XVE does not containOct-3/4 in vivo (Fig. 3C–G). By con-trast, two epiblast-derived stem celllines, EG cells (Matsui et al., 1992)and EpiSC (Brons et al., 2007; Tesaret al., 2007), both of which are pluri-potent, express Oct-3/4 and reflectOct-3/4’s status in vivo in the earlypost-implantation epiblast (Scholer etal., 1990). Human amniotic stem cells,both those that have been isolatedfrom term amnion (Miki et al., 2005)and those from amniotic fluid (DeCoppi et al., 2007), express Oct-3/4(Figs. 2F,G, 3A–H).

Correlation of Oct-3/4 in epiblast-

derived stem cell lines and corre-sponding tissues in vivo suggests that,whatever the tissue, as long as it ex-presses Oct-3/4, unique cell linesmight be derived from it. Two promis-ing candidates are chorionic meso-derm and the allantois. Between 4-and 8-s, these unite and contribute tothe nascent chorio-allantoic placenta(Downs and Gardner, 1995), a majorsite of definitive hematopoiesis (Gekaset al., 2005; Otterbach and Dzierzak,2005). The source of placental hema-topoietic cells has not been identified,but both the chorion and allantois ex-hibit definitive hematopoietic poten-tial (Zeigler et al., 2006). Given therobust levels of Oct-3/4 in chorionicmesoderm and the allantois (Figs. 2G,3A), their survival in culture (Downset al., 2001; Zeigler et al., 2006), andthe presence of pluripotent cellswithin the proximal allantois (Downsand Harmann, 1997), chorionic meso-derm and the allantois may be idealtissues for deriving pluripotent, vas-cular, and/or hematopoietic cell lines.

Do Allantoic Oct-3/4-PositiveCells Contribute to theGerm Line?

A number of studies have suggestedthat the allantois contains PGCs (Chi-quoine, 1954; Ozdzenski, 1967; Gins-burg et al., 1990). Yet, the PGC found-ing population, which emerges duringearly gastrulation, is thought to besmall (Lawson and Hage, 1994). Onthat basis, Oct-3/4’s widespread abun-dance in the nascent allantois makesit unlikely that all allantoic Oct-3/4-positive cells are primordial germ cells(Scholer et al., 1990). Nevertheless,given the number of multi-lineage de-scendents to which Oct-3/4 progenitorpopulations contribute, the largenumber of Oct-3/4-expressing celltypes, and Oct’s localization to germcells in the genital ridges (Scholer etal., 1990), it is tantalizing to speculatethat a subpopulation of allantoic cellscontributes to the germ line.

Results of studies here suggest var-ious routes by which allantoic PGCs, ifthey exist, might become part of thehindgut. First, AX Oct-3/4-positivecells may originate within the allan-tois and, from there, become part ofthe hindgut by tissue translocation, assuggested in the “Contribution of Oct-

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3/4-Positive Cells to Gut Endoderm”section. Although orthotopic graftingexperiments failed to identify donorallantoic cells in the hindgut, donorallantoic cells had been introducedinto the host’s proximal allantois di-rectly through adjacent AX, undoubt-edly damaging it and pre-empting as-sessment of its contribution todefinitive endoderm (Downs and Har-mann, 1997).

Alternatively, allantoic PGCs mightenter the blood stream, as allantoicOct-3/4-positive cells are closely asso-ciated with the allantoic blood vessels(Fig. 6F). Although blood-borne deliv-ery of germ cells to the gonads has noprecedent in mammals, in mouse tes-tes, time lapse imaging has recentlyrevealed that spermatogonia are lo-cated near blood vessels (Yoshida etal., 2007). Moreover, chick PGCs hometo the gonadal ridges via the blood-stream (Simon, 1960; Fujimoto et al.,1976).

Lastly, allantoic PGCs may becomepart of the fetal hindgut by direct as-sociation between the allantois andhindgut (Fig. 6E). Fate mapping prox-imal allantoic cells failed to demon-strate contribution to embryonic tis-sues (Downs and Harmann, 1997).Rather, all descendents were foundwithin the allantois, suggesting that,once inside this organ, movement ofallantoic cells is from proximal-to-dis-tal. However, grafting into the allan-tois at a level too far removed from, ordistal to, the prospective hindgut toassociate with gut endoderm, cannotbe ruled out.

Conclusions

Together, these findings present acomprehensive profile of Oct-3/4 dur-ing mouse gastrulation. Further, theysuggest new roles for Oct-3/4 in manycell types, and call into question Oct-3/4’s use as a global “marker” of plu-ripotency. It is anticipated that thisdevelopmental blueprint of Oct-3/4’swhereabouts during early murinepost-implantation development willguide future investigations into Oct-3/4’s manifold and, undoubtedly, com-plex, roles in differentiation of the epi-blast during specific windows ofgastrulation.

EXPERIMENTALPROCEDURES

All animals were treated in accor-dance with Public Health Service(PHS) Policy on Humane Care andUse of Laboratory Animals (PublicLaw 99-158) as enforced by the Uni-versity of Wisconsin-Madison. For allexperiments, n � 3 specimens for eachstage, unless otherwise noted.

Mouse Strains, AnimalHusbandry, Dissection, andStaging

The F2 generation of matings betweenthe inbred hybrid strain B6CBAF1/J(Jackson Laboratories) was used(Downs, 2007). Flk-1/Oct-3/4 co-local-ization studies employed a maleKdrtm1Jrt mouse (hereafter referred toas Flk-1lacZ) (Shalaby et al., 1995) aspreviously described (Inman andDowns, 2006b). Animals were main-tained under a 12-hr light/dark cycle(lights out 13:00), and estrus selec-tion, dissection, and embryo stagingwere as previously summarized(Downs and Davies, 1993; Downs andGardner, 1995; Downs, 2007). Briefly,pregnant females were killed by cervi-cal dislocation, conceptuses were dis-sected away from decidual tissues,Reichert’s membrane and associatedtrophoblast were reflected, and stagesand their equivalent approximate dpcwere categorized as follows: early,mid-, and late streak (ES, MS, LS)stages, �6.75–7.0 dpc; no allantoicbud (OB, 7.0–7.25 dpc); early and lateallantoic bud (EB, LB) stages, �7.25–7.5 dpc; early and late headfold (EHF,LHF) stages, �7.75–8.0 dpc. Thereaf-ter, staging was by numbers of somitepairs (1–2-s, 8.0–8.25 dpc; 2–4-s, 8.25dpc; 4–6-s, 8.25–8.5 dpc; 6–8-s, 8.5–8.75 dpc; 9–16-s, 8.75–9.25 dpc).

X-Gal Staining andImmunohistochemistry

Flk-1lacZ conceptuses were fixed in 4%paraformaldehyde (2 hr, 4°C) andstained for X-gal activity as previouslydescribed (Downs and Harmann,1997), but for 6 hr only, after whichspecimens were rinsed in phosphate-buffered saline (PBS, Sigma) and de-hydrated in increasing methanols asdescribed below prior to immunostain-ing.

Immunostaining Bouin’s- and 4%paraformaldehyde-fixed and sectionedspecimens was as previously de-scribed (Inman and Downs, 2006a) us-ing primary antibody to Oct-3/4 (N-19,goat polyclonal SC-8628, Santa CruzBiotechnologies, Santa Cruz, CA) atdilutions of 1/67, 1/100, and 1/250 for1.5 and 3.0 hr at room temperature,and 18 hr at 4°C. For whole mountimmunohistochemistry, all concep-tuses were fixed in 4% paraformalde-hyde (2 hr, 4°C), and rinsed in phos-phate-buffered saline (PBS, Sigma),followed by dehydration in an increas-ing series of methanols, and indefinitestorage at �20°C in absolute metha-nol. Just prior to immunostaining, theyolk sac was opened up in all speci-mens � OB stage by gentle tearingwith a forceps. For immunostaining,all steps were carried out on a rockingplatform at room temperature, withthe exception that primary and sec-ondary antibody binding were carriedout at 4°C on the rocker. Endogenoushydrogen peroxidase activity waseliminated with 5% hydrogen peroxi-dase/methanol for 5 hr. This was fol-lowed by blocking donkey non-specificantigen sites for 2 hr in PBS contain-ing 5% donkey serum (Chemicon) and0.1% Triton-X (“PBSST”). Specimenswere incubated overnight at 4°C inprimary antibody against Oct-3/4 (1/100 dilution in PBSST), and the nextday, they were washed 5 times in PB-SST for a total period of 5 hr, followedby application of secondary antibody(1/500 dilution in PBSST; biotinylateddonkey anti-goat IgG, SC-2042; SantaCruz Biotechnologies, Santa Cruz,CA) and incubation overnight at 4°C.After 5 hr of rinsing in PBSST, speci-mens were incubated for 3 hr in ready-to-use ABC reagent (PK-7100, VectorLabs, Burlingame, CA), washed 3times during 1.5 hr in PBSST, thentwice during 20 min in PBT, in whichbovine serum albumen (0.02%, Sigma,A-4378) replaced the donkey serum.The antibody complex was visualizedwith diaminobenzoate chromagen(DAKO Corporation, Carpinteria,CA) for 5 min at room temperature,after which specimens were fixedagain in paraformaldehyde at 4°Covernight, oriented transversely,sagitally, or frontally, and embed-ded, after which they were sectionedat a thickness of 6 �m, dewaxed,

OCT-3/4’S EXPRESSION PROFILE IN MOUSE 473

counterstained in hematoxylin, cov-erslipped, and examined in the com-pound microscope. Control experi-ments included elimination ofantibody, and pre-binding the Oct-3/4 antibody with Oct-3/4 peptide(SC-2042p, Santa Cruz Biotechnolo-gies) for 10 hr at 4°C.

Measuring the AllantoicOct-3/4 Domain

Sagital and transverse orientations ofthe allantois were used to measureallantoic length and the length of theOct-3/4 domain, as briefly described inthe text. Sagital measurements weremade at the EB and LB stages, usingprevious criteria that defined the baseof the allantois as its site of insertioninto the amnion and visceral yolk sacat the headfold stage (Downs and Har-mann, 1997) (Fig. 3A,B). However,this criterion omitted the Oct-3/4-pos-itive region adjacent to the AX (Fig.3A,B). Nevertheless, inclusion of thisregion would not alter the length ofthe Oct-3/4 domain for these EB andLB stages, which extended through-out the entire allantoic projection (Fig.4A,B). By the EHF stage, the length ofthe Oct-3/4 domain began to shorten,from which time on calculations weremade in transverse sections. At thisEHF and all subsequent stages, thefirst section of the base of the allantoiswas distinguished from the boundarywith the primitive streak by the pre-cipitous decrease in left-right width ofthe base of the allantois relative tomeasurements of the two previoussections (e.g., Fig. 1F–I). Not only didtransverse orientations allow detailedviews of the intensity of all Oct-3/4cells at every allantoic level, but theyprovided a means for appraising itsdomain with respect to anteroposte-rior, dorsal-ventral, and left-right co-ordinates, and accurate counts of AX-containing Oct-3/4 cells. Transversemeasurements included the region ad-jacent to the AX (Fig. 3C). The“straightness” of transverse sectionswas judged with respect to the AX,whose shape was transitional betweensquamous (EVE, embryonic visceralendoderm in the embryonic region),and cuboidal (XVE, extraembryonicvisceral endoderm in the visceral yolksac) (Fig. 3C), and generally devoid oflarge vesicles (not shown). The num-

ber of sections containing intenselystained Oct-3/4 cells was determinedcomplete if the next two sections didnot contain darkly stained cells, illus-trated in a sagital section (Fig. 3C). Noeffort was made in this study to esti-mate the extent of tissue shrinkageafter histological preparation; thus,all measurements were based oncounting the number of uninterruptedserial 6-�m-thickness histological sec-tions.

ACKNOWLEDGMENTSThe author is grateful to LandenRentmeister for initial technical assis-tance with Figure 1A, to Dr. Val Wil-son, Dr. Kate Storey, and Dr. ClaudioStern for valuable discussions on thenode’s architecture and stem cell po-tential, and the National Institutes ofHealth (RO1HD042706) for support.

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