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Keywords:

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

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

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 distinct epiblast-derived embryonic and extraembryonic tissues. In the allantois, distal-to-proximal diminution in the Oct-3/4 domain over time and co-localization with Flk-1 in angioblasts accorded with a role in vascular differentiation and the presence of a stem cell reservoir. In addition, visceral endoderm exhibited a dynamic salt-and-pepper distribution, which, combined with previous results of fate mapping and gene expression, suggested that Oct-3/4 is involved in the genesis of definitive endoderm. By 8-s, Oct-3/4 was globally 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 in development than have been previously appreciated. Developmental Dynamics 237:464–475, 2008. © 2008 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

During embryonic development, cell fate is regulated by transcription factors that act as molecular controls to activate or repress specific batteries of gene expression. Oct-3/4, encoded by Pou5f1, is thought to be one of the earliest transcription factors involved in differentiation in the mammalian conceptus (reviewed in Stefanovic and Pucéat,2007).

Oct-3/4 is associated with toti- and pluripotent cells during early pre-implantation development in both mice and humans. In these species, Oct-3/4 was found as maternal and embryonic protein in the unfertilized egg, zygote, and the zygote's descendents through the blastocyst stage (Palmieri et al.,1994; Cauffman et al.,2006). Once the blastocyst formed, Oct-3/4 was down-regulated in trophectoderm, thereafter being found exclusively in the blastocyst's inner cell mass (ICM) (Palmieri et al.,1994; Cauffman et al.,2006).

Manipulation of murine embryonic stem (ES) cells provided compelling evidence that Oct-3/4 may control, in a dose-dependent manner, cell fate as the ICM segregates into epiblast and primitive endoderm (Palmieri et al.,1994; Niwa et al.,2000). Surprisingly, for so important a gene product in potency and differentiation, the comprehensive whereabouts of Oct-3/4 in ICM derivatives beyond implantation are not known.

Gastrulation begins at about 6.5 dpc, and is the time when the primitive streak, or embryonic antero-posterior (A-P) axis, is established and the three primary germ layers, ectoderm, mesoderm, and endoderm, systematically segregate from the epiblast. Over a 48-hr period, spatial coordinates established by the streak direct differentiation of the germ layers, and together these components lay down the basic body plan of the fetus (Beddington,1983).

In light of the importance of gastrulation and Oct-3/4's role in regulating developmental potency, I have investigated the whereabouts of Oct-3/4 protein in the mouse conceptus. Previous localization to the gastrula between 6.0 and 11.0 dpc painted broad brushstrokes of Oct-3/4 mRNA, describing it in epiblast, neurectoderm, and the base of the allantois, where expression was reputed to be in primordial germ cells (PGCs) (Scholer et al.,1990). Findings here reveal that the number of Oct-3/4-expressing sites during gastrulation far exceeded those previously reported. As the primary germ layers were deployed from the epiblast, they, and subsequently their own progeny, exhibited robust levels of Oct-3/4. Over time, Oct-3/4 diminished, leaving just two clear positive cell populations, putative PGCs, and hitherto unclassified allantoic cells.

These results provide a comprehensive and fundamental spatiotemporal blueprint for evaluating the significance of Oct-3/4's gene activity in differentiating cells and tissues. Further, they point to tissues that may, on the basis of Oct-3/4 expression, be used to create unique stem cell lines.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Immunostaining Whole Mount Prepared Specimens Produced Superior Oct-3/4 Signal

Several previous studies from my laboratory revealed variability in the results of immunostaining according to the type of fixative used. For example, AHNAK and T signals in sections of Bouin's-fixed specimens exceeded those obtained in paraformaldehyde (Downs et al.,2002; Inman and Downs,2006a). Thus, in this study, I compared the robustness of Oct-3/4 in Bouin's- or paraformaldehyde-fixed and sectioned material with that in whole mounted specimens fixed in 4% paraformaldehyde/methanol followed by immunostaining and sectioning.

Immunostaining sections of material fixed in paraformaldehyde yielded no Oct-3/4 signal (see Experimental Procedures section; data not shown). By contrast, Bouin's-fixed specimens exhibited an Oct-3/4 signal that, whilst localized to the same sites as those in whole mount preparations, was nevertheless inferior in intensity and more diffuse than that in whole mount preparations (compare Fig. 1A,B). Subsequently, for each stage examined here, all material was immunostained in whole mounted specimens prior to embedding and sectional analysis.

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Figure 1. Comparison of Oct-3/4 immunohistochemistry; the posterior primitive streak/allantois boundary in transverse orientation. In this and all other panels, histological sections were lightly counterstained with hematoxylin (purple color); Oct-3/4 protein is indicated by brown color. In A, both sets of orthogonal arrows indicate that, unless otherwise noted, anterior (a) is on the left, and posterior (p) is on the right in all panels 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 provide similar distal/proximal coordinates for the extraembryonic portion above the amnion. In whole mount specimens, the anterior yolk sac was opened prior to immunostaining. A–E: Sagital orientation. F–G: Transverse orientation. I: Frontal orientation. A: EHF, Bouin's fixed, sectioned, and immunostained with anti-Oct-3/4. B–I: Whole mount immunostained preparations followed by sectioning. B–E: From the same control experiment. B: EHF, 4% paraformaldehyde/methanol whole mount immunohistochemistry with anti-Oct-3/4. C: EHF, anti-Oct-3/4 was placed at 4°C for 10 hr prior to use. D: EHF, anti-Oct-3/4 + control Oct-3/4 peptide, 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) is non-specific. F–H: 2-s, three transverse sections illustrating the posterior 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-μm levels at which the sections in F–H were taken, with the shortest line at the top corresponding to the base of the allantois in H, the middle line to the al/pps in G, and the lower line to the pps in F, the latter two of which are of similar length (see text for details). Scale bar in E = 200 μm (A–E); in H = 50 μm (F–H), in I = 50 μm (I). + Ab, presence of anti-Oct-3/4 antibody; −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, headfolds; pps, posterior primitive streak.

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Pre-binding the Oct-3/4 antibody with control peptide for 10 hr at 4°C as well as immunostaining in the absence of antibody to Oct-3/4 revealed specificity for all anatomical sites except trophoblast giant cells and parietal endoderm (Fig. 1C–E, and data not shown). In all sites identified here, and at all stages, Oct-3/4 localized solely to nuclei.

Early Gastrula: Streak Stages (∼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 tissues were the epiblast, primitive streak, embryonic and extraembryonic mesoderm, and embryonic visceral endoderm (EVE) (Fig. 2A–C). The Oct-3/4 EVE domain extended circumferentially around the egg cylinder, from the anterior level of the primitive streak to the distal tip of the egg cylinder (Fig. 2A–C).

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Figure 2. Oct-3/4 in streak and neural plate stages. A: MS, sagital section. Thin horizontal line through the posterior epiblast and overlying EVE indicates the anteriormost limit of the primitive streak at this stage. Anterior and posterior arrowheads indicate the extent of Oct-3/4 cells in EVE. B,C: MS, transverse sections 54 (B) and 30 (C) μm, respectively, from the distal embryonic tip of a second conceptus. White asterisk in B, primitive streak; in C, the streak is not present. Arrowheads indicate examples of distinct Oct-3/4-positive cells within the EVE. D: LS, sagital section. Arrowheads as in A. The streak has now reached its full length, and its anterior end has condensed into the node (arrow). E: OB, sagital section of anterior half and distal tip, including the node (arrow). Anterior arrowhead 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 its anterior length is similar to that of the epiblast. Also note that lateral plate mesoderm (lpm) between the EVE and epiblast is mottled, exhibiting positive and negative Oct-3/4 cells, whilst paraxial mesoderm (pm) is homogeneously 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 exocoelomic 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-positive cells, whose intensity was diminishing with respect to more posterior notochordal 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 the primitive streak (asterisk). Some notochordal cells (nt), located opposite the streak in this section, still exhibited Oct-3/4. Arrowheads as in I. Scale bar in J = 50 μm (A, D–I); 75 μm (J); in C = 50 μm (B, C). e, epiblast; lpm, lateral plate mesoderm; np, neural plate; nt, notochord. pm, paraxial mesoderm; x, exocoelomic cavity; xm, extraembryonic mesoderm.

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At the Late Streak (LS) stage (∼7.0 dpc), the only new Oct-3/4-positive structure was the node as it emerged at the anterior end of the primitive streak (Fig. 2D). Oct-3/4-positive EVE cells were now associated with the full circumference of paraxial and lateral plate mesoderm. With the exception of extraembryonic mesoderm, all extraembryonic tissues were negative during 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 notochord was elongating through the midline from its origin in the node, and contained intensely positive Oct-3/4 cells (Fig. 2E). On either side of this structure, EVE cells were also positive, overlying prospective paraxial and lateral plate mesoderm (not shown for this stage, but see the LB stage, Fig. 2I,J, below). Posterior lateral plate mesoderm and nascent prospective paraxial mesoderm themselves were intensely positive (compare Fig. 2E,F). By contrast, anterior-half lateral plate mesoderm was of mixed intensity, varying from slightly positive to negative (Fig. 2E).

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

Neural Plate/Allantoic Bud Stages (∼7.25–7.5 dpc)

At both the early (EB, ∼7.25 dpc) and late allantoic bud (LB, ∼∼7.5 dpc) stages, Oct-3/4 patterns were similar (Figs. 2H–J, 3A,B). In the embryonic region, Oct-3/4 was still robust in the epiblast, primitive streak, and node (Fig. 2H). The notochord had reached its full length, but anterior cells were either negative, or less intensely positive than posterior ones (Fig. 2H).

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Figure 3. Oct-3/4 in the allantois and allantoic vasculature, associated visceral endoderm, and hindgut endoderm. A: EB, sagital profile. B: LB, sagital profile. Oblique lines in A,B connect the site of insertion of the allantois into the amnion and yolk sac, and define the base of the allantois as previously described (Downs and Harmann,1997), and the proximal level from which the base to the distal tip was measured for Figure 4. C: EHF, sagital profile. Arrowheads indicate Oct-3/4-positive cells in allantois-associated visceral endoderm (ax) (top arrowhead) and EVE (eve) overlying the posterior primitive streak (bottom arrowhead). The horizontal line indicates the level above which the length of the Oct-3/4 domain was measured in transverse sections from this stage onward; note that it includes those Oct-3/4 cells adjacent to the AX. For further details, see text and Experimental Procedures section. D: 1-s, transverse section. Oct-3/4 positive amniotic ectoderm (arrows on am) and AX (arrowheads) are particularly clear in this section. E: 5-s, semi-oblique sagital/frontal section. The thin line extends across the boundary between the base of the allantois and the embryonic region, as previously defined in A,B, above; thus, the Oct-3/4-positive portion of the allantois and Oct-3/4-positive hindgut are physically continuous. Arrowheads indicate Oct-3/4-positive cells in AX (top arrowhead) and in EVE overlying the embryonic hindgut (bottom arrowhead). F: 5-s, sagital section revealing the association between Oct-3/4-positive allantois, AX cells, and the primary umbilical vessel (asterisks). G–J: 2-s. Consecutive series of transverse sections at specific levels through an X-gal-stained Flk/LacZ allantois, the angioblasts of which are royal blue (purplish color is hematoxylin), immunostained with anti-Oct-3/4 (brown). In all panels, arrows indicate Flk/Oct-3/4-positive cells; arrowheads in G indicate Oct-3/4-positive cells in the AX; 18 (G), 60 (H), and 72 (I) μm from the boundary with the posterior primitive streak. Insets (I) are examples of X-gal-positive cells from this section either co-stained with Oct-3/4 (top inset), or negative for Oct-3/4 (bottom inset). J: 150 μm from the streak boundary. Scale bar in F = 50 μm (A–F), in J = 50 μm (G–J). hg, hindgut; ps, primitive streak; xve, extraembryonic visceral endoderm.

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Paraxial mesoderm was darkly stained (Fig. 2I), but only posterior lateral plate mesoderm exhibited homogeneous Oct-3/4 (Fig. 2J). Oct-3/4-positive EVE was associated with paraxial and lateral plate mesoderm (Fig. 2I,J) and, in a minority of specimens (EB stage, 20%, N = 5; LB stage, 40%, N = 5; not shown), began to extend to posterior primitive streak-associated EVE. The neural plate contained Oct-3/4 (Fig. 2I).

In the extraembryonic region, exocoelomic mesoderm was still positive (Fig. 3A,B), but nascent yolk sac blood islands did not exhibit Oct-3/4 at this, or at any other, time (not shown). The allantoic bud had appeared and, during the bud stages, its base was both continuous with the underlying primitive streak and adherent to overlying extraembryonic visceral endoderm. Henceforth, allantois-associated extraembryonic visceral endoderm shall be referred to as “AX.” Bud-stage allantoises were examined in sagital orientation, the base of the allantois taken as its site of insertion into the visceral yolk sac and amnion, as previously described (Downs and Harmann,1997) (Fig. 3A,B; also see Experimental Procedures section). Both inner and outer allantoic cell populations contained Oct-3/4 along the entire allantoic length (25–87 μm) (Figs. 3A,B, 4A,B). The AX itself was negative for Oct-3/4 at this time (Fig. 4C). All other extraembryonic cells were negative (data not shown).

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

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Headfold Stages (∼7.75–8.0 dpc)

At the early headfold (EHF) stage (∼7.75 dpc), the newly formed anterior neurectoderm and surface ectoderm exhibited Oct-3/4 (Fig. 1B,C). In the node, Oct-3/4 localized to all but its ventral anterior aspect, where Oct 3/4 was now undetectable (Fig. 5A,B). The EVE pattern of Oct-3/4 had also shifted, and was now confined to the posterior half of the embryo, including the posterior primitive streak. All other posterior embryonic cells and tissues were positive.

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Figure 5. Oct-3/4 in the node and associated EVE. A: EHF, sagital section through the node. Arrow points 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 conceptus through the Oct-3/4-positive posterior crown (arrowhead). C: 1-s, sagital section. Arrow and arrowhead 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 intimately associated 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 μm. E: Anterior ventral node (arrow) was negative for Oct-3/4, whilst the dorsal node (asterisk here and in all subsequent panels) 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 contains mixed Oct-3/4-negative and -positive cells; the adjacent EVE was Oct-3/4-positive. G: The posterior ventral node (arrowhead) and posterior dorsal node (asterisk). Adjacent EVE was deeply positive. H: The node has disappeared, and the putative primitive streak is now physically continuous with overlying Oct-3/4-positive EVE (thin arrow). Scale bar in H = 50 μm (E–H), 39 μm (A, C), 28 μm (B, D).

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In the extraembryonic region, Oct-3/4 was found predominantly in inner, and occasionally outer, associated cells of the allantois (Fig. 3C). Allantoises were now analyzed for their content of Oct-3/4 in transverse orientation (for details, see Experimental Procedures section); by the EHF stage, most (83.3%) were consistently directed toward the chorion. By the LHF stage, all allantoises were consistently pitched toward the chorion.

At the EHF stage, the length of the allantois had exceeded 100 μm in 83.3% of specimens (Fig. 4A) and, in these, Oct's distal domain was shortening relative to the total allantoic length, becoming confined to the mid- and proximal thirds (Figs. 3C, 4B). In transverse sections, the Oct-3/4 domain appeared symmetric about the allantois's putative A-P axis, but asymmetric with respect to its dorsal-ventral (D-V) one (data not shown for the headfold stages, but see 1-s stage, Fig. 3D). The more ventral portion of the allantoic Oct-3/4 domain was closely apposed to the overlying AX, where Oct-3/4 was observed for the first time at the EHF stage (Figs. 3C, 4C).

In the remaining 16.7% of EHF-stage material (N = 1, not included in Fig. 4 calculations; see Experimental Procedures section), the length of the allantois was 80 μm, similar to that found at the LB stage (Fig. 4A). In this specimen, the Oct-3/4 domain occupied the entire length of the allantois, similar to the earlier EB and LB stages. Thus, 87–100 μm must be the critical allantoic length at which reduction in the Oct-3/4 domain occurs. By contrast with the previous stages, however (Fig. 4B), the AX in this EHF-stage specimen exhibited six Oct-3/4-positive cells, suggesting that emergence of AX Oct-3/4 cells is stage-, rather than length-, dependent.

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

The posterior aspect of the node remained positive through 5-s (Fig. 5C,D). A series of transversely oriented specimens highlighted the intimate association between the posterior ventral portion of the node, and EVE (Fig. 5E–H), both of which contained Oct-3/4 (Fig. 5D–G). Furthermore, Oct-3/4-positive EVE was confined to that overlying the embryonic primitive streak.

Anterior and posterior neurectoderm exhibited robust levels of Oct-3/4 until 5-s (Fig. 6A). By 6-s, levels of Oct-3/4 diminished, with anterior neurectoderm losing this protein in advance of posterior neurectoderm (Fig. 6C). Precocious loss of anterior Oct-3/4 was particularly striking at 7-s (Fig. 6D). As somites emerged from paraxial mesoderm, they were initially slightly positive, but, with age, their intensity lessened with rostral-to-caudal directionality (Fig. 6A,B). Nascent hindgut endoderm, located beneath the allantois, contained Oct-3/4 (Fig. 3E).

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Figure 6. Diminution of Oct-3/4 in embryonic and extraembryonic tissues. A: 5-s, sagital profile illustrates that, with the exception of neurectoderm and the amnion, epiblast-derived anterior tissues do not exhibit Oct-3/4 whilst posterior embryonic structures, the allantois and amnion, are highly positive. Arrowhead points to Oct-3/4-positive cells in AX overlying the primary umbilical vessel (asterisk). B: 5.-s, sagital section through somites. The most recently formed posterior somite (right arrow) contains more Oct-3/4 than an older anterior one (left arrow). C: 6-s, transverse section showing reduced Oct-3/4 in anterior neurectoderm (an) by comparison with posterior neurectoderm (pn). D: 7-s, transverse section showing further diminution in Oct-3/4 in anterior neurectoderm. 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 the embryo. Putative PGCs (arrowheads), ventral aspect of the hindgut. F,G: 10-s, oblique sagital sections 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 bar in H = 50 μm (B, H); 86 μm (C, D); 100 μm (E, G); 200 μm (F). Scale bar in A = 200 μm (A). bi, blood islands; fg, foregut; ht, heart; ne, neurectoderm; s, somite; se, surface ectoderm.

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In the extraembryonic region, amniotic ectoderm was positive through 5-s (Fig. 3A–F) whist the mesodermal lining of the exocoleomic cavity gradually became Oct-3/4-negative. The proximal allantoic Oct-3/4 domain was associated with the AX (Fig. 3C), hindgut (Fig. 3E), and the umbilical vasculature (Fig. 3E,F). Until 6-s, allantoic contact with the AX persisted (Fig. 3C–F, and not shown), with the AX's Oct-3/4-positive cell content highest at 2- and 4-s (Fig. 4C).

Through 3-s, the absolute length of the Oct-3/4 domain increased (Fig. 4A). However, despite increasing allantoic length (Fig. 4A), the proportional length of the Oct-3/4 domain in the mid- and proximal allantoic regions was fairly constant through 3-s, after which expression diminished with increasing embryonic age, becoming confined to the proximal quarter of the allantois's full length by 6-s (Fig. 4B). Persistence of the proximal Oct-3/4 allantoic domain suggested the presence of a stem cell reservoir, a notion supported by previous results that revealed pluripotent cells in this region (Downs and Harmann,1997). Moreover, distal-to-proximal diminution in the Oct-3/4 domain relative to the increased length of the allantois was reminiscent of allantoic vascularization, which occurs with distal-to-proximal polarity throughout this period (Downs et al.,1998). Lastly, Oct-3/4 was associated with the allantoic vasculature (Fig. 3E,F). Together, these observations suggest that Oct-3/4 is involved in allantoic vasculogenesis.

To discover Oct's potential role in allantoic vascularization, Flk-1-containing allantoic angioblasts (Downs et al.,1998) were co-stained for Oct-3/4 in Flk-promoter-driven LacZ transgenic conceptuses. At EHF and 4-s stages (N ≥ 3), the average length of the allantois and Oct-3/4 domain fell 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), demonstrating that the presence of the lacZ transgene had no effect on allantoic growth. Moreover, the average length of the Oct-3/4 domain was similar in both genetic backgrounds (EHF: P = 0.28; 4-s: P = 0.22). During the entire period examined (EHF-5-s stages), doubly positive Oct-3/4/Flk-1 cells were identified (e.g., Fig. 3G–J). All Oct-3/4 cells bearing Flk-1 were mildly, 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 few cell types, including the putative germ cell population, some clusters of hitherto unidentified cells throughout the allantois, and an occasional surface ectodermal cell (Fig. 6E). Within the hindgut, Oct-3/4-positive cells were localized to the ventral, rather than dorsal, region (Fig. 6D,E). Specimens examined at 10-, 12-, 14-, and 16-s pairs exhibited similar patterns and intensity of staining, with no new positive cell types emerging (Fig. 6F–H). Within the allantois, Oct-3/4-positive cells were round and generally clustered.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Summary of Oct-3/4 in the Mouse Gastrula

Results here demonstrate Oct-3/4's presence as an uninterrupted continuum in mouse epiblast and its progeny tissue as the latter emerge and mature within the gastrula. Epiblast, the primitive streak, node, notochord, nascent embryonic mesoderm, neural plate, mid- and distal EVE, nascent extraembryonic mesoderm, and amniotic ectoderm exhibited intensely stained Oct-3/4 cells during pre-allantoic bud stages. By bud stages, new positive structures, neurectoderm, surface ectoderm, proximal posterior EVE, and the allantois emerged. Oct-3/4 diminished in the notochord and anterior lateral plate mesoderm. At headfold stages, only the AX emerged as a new Oct-3/4-positive tissue. Others were losing Oct-3/4, including the anterior node, anterior EVE, and distal allantois. During 1–7-s stages, the only new positive structure was the hindgut; Oct-3/4 was down-regulated in paraxial mesoderm-derived somites, the walls of the exocoelom, and the distal-to-mid-allantoic regions. In specific structures, Oct-3/4 became confined to the posterior, or caudal node, that EVE overlying the caudal node and embryonic primitive streak, the AX, and the core of the proximal allantois. Between 8–16-s stages, Oct-3/4 was almost globally extinguished, being detectable only in putative PGCs of the hindgut, an occasional surface ectodermal cell, and small cell clusters in the allantois.

Oct-3/4 was limited to nuclei. During ES, MS, and LS stages, these were uniformly dark in positive tissues. Between the OB-8-s stages, Oct-3/4's intensity ranged from almost black to light brown in most tissues. However, for reasons that are not clear, amongst positive tissues, only the epiblast, primitive streak, neurectoderm, posterior embryonic mesoderm, and amniotic ectoderm seemed uniformly positive throughout their periods of expression. Two tissue subdomains, the core domain within the base of the allantois and the caudal node, also seemed homogeneously positive at the outset of their expression. At >8-s, staining was either intensely dark or undetectable.

Although the nature of later-stage allantoic Oct-3/4 cells is not known, their shape and clustering were reminiscent of yolk sac blood islands (Haar and Ackerman,1971). Patency of the allantoic vasculature with the yolk sac and fetal arterial systems begins at the 4-s stage (Downs et al.,1998), yet it is unlikely that extrinsic hematopoietic cells entered the allantois via the vasculature. Not only was Oct-3/4 undetectable in yolk sac blood islands/circulating blood cells, but allantoic clusters were not contained within blood vessels. Rather, allantoic Oct-3/4 cells may represent an intrinsic and, possibly, emerging definitive hematopoietic population (Zeigler et al.,2006).

Previous results of fate mapping suggest that gradual extinction of Oct-3/4 corresponds with increasing cell age and differentiation. This was best illustrated in the notochord/foregut (Fig. 2E, H) (Beddington,1994; Tam et al.,2004), paraxial mesoderm/somites (Fig. 6A,B) (Tam and Beddington,1987), lateral plate 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 that are not yet understood, near-global extinction of Oct-3/4 beyond 7-s (Fig. 6E) demonstrates that Oct-3/4 is not required for growth and morphogenesis of individual organ systems (Kaufman,1992).

Oct-3/4 Plays a Major Role in Building the Umbilical Vasculature

Oct-3/4 triggers expression of mesodermal and cardiac-specific genes through Smad2/4 in embryonic stem (ES) cells, driving the latter toward differentiation into cardiomyocytes (Zeineddine et al.,2006). In accord with this conclusion in vitro, Oct-3/4 was highly expressed in prospective heart mesoderm, which emerges from the primitive streak at the MS stage (Kinder et al.,1999). Thus, results of this study support a role in vivo for Oct-3/4 in heart development.

In addition, results revealed that Oct-3/4 is involved in vascularization of the allantois. The allantois, which is the precursor tissue of the umbilical cord, undergoes de novo vasculogenesis (Downs et al.,1998; Drake and Fleming,2000). Flk-1-positive angioblasts were initially most abundant in the distal allantois at the LB/EHF stages (Downs et al.,1998), where allantoic cells are oldest (Downs and Harmann,1997; Kinder et al.,1999).With distal-to-proximal directionality, Flk-1-positive angioblasts emerged down the length of the allantois such that, by 4–6-s, a primary Flk-1-positive umbilical vasculature was formed (Downs et al.,1998,2004; Inman and Downs,2006b, 2007). These observations led to a model of polarized allantoic vascularization (Downs et al.,2004). Intriguingly, as Flk-1 is acquired with distal-to-proximal kinetics (Downs et al.,1998), Oct-3/4 diminished with the same directionality (Fig. 4A,B). This correlation lasted throughout vasculogenesis, with many non-robustly-stained Oct-3/4 cells exhibiting Flk-1. Moreover, persistence of Oct-3/4 within the proximal core domain strongly argues for this region as a stem cell reservoir for building the differentiating allantoic vasculature. This region contains Brachyury, or T (Inman and Downs,2006a), which, as recently demonstrated, is required for allantoic vascularization and patterning (Inman and Downs,2006b) On the basis of these findings, I conclude that Oct-3/4 plays a significant role in formation of the umbilical vasculature.

Contribution of Oct-3/4-Positive Cells to Gut Endoderm

The origin of definitive gut endoderm is complex, but involves intercalation of streak- (Lawson et al.,1986,1991; Lawson and Pedersen,1987) and epiblast- (Tam and Beddington,1992) derived definitive endoderm into the EVE. As a result, gene expression in this cell layer might be expected, in certain cases, to exhibit salt-and-pepper distribution. Oct-3/4-positive cells were located amongst unstained EVE cells through 6-s (Fig. 7). Recently, the chick homolog, PouV/Oct-3/4, exhibited similar expression patterns in the hypoblast, the chick equivalent of mouse visceral endoderm (Lavial et al.,2007). Results of fate mapping, gene expression, and ablation studies (reviewed in Lewis and Tam,2006) demonstrated that genesis of definitive gut endoderm involves a variety of gene activities and morphogenetic movements, as descendents of the bud-stage node and three EVE regions, “posterior-distal,” “posterior-middle,” and “posterior-proximal,” became part of the embryonic gut (Tam et al.,2004). In the present study, all of these exhibited Oct-3/4 (e.g., Figs. 2H–J, 7). Moreover, Oct-3/4's expression pattern in EVE was uncannily similar to that of Cerberus between the LS and LB stages (Fig. 7B) (Lewis and Tam,2006). Together, these observations support a role for Oct-3/4 in development of definitive endoderm.

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Figure 7. Summary of Oct-3/4 in visceral endoderm over time. Schematic diagram of the embryonic portion of the egg cylinder, proximal allantois, and AX. 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 visceral endoderm (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 (*, and heavy line), the notochord (nt), and EVE overlying the primitive streak at the level of lateral plate mesoderm (lpm) and paraxial mesoderm (pm). The circles 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 this posterior region (see text for details). *', *”, and *”' are the “posterior-distal,” “posterior-middle,” and “posterior-proximal” regions fate-mapped by Tam et 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 region overlying 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 the remainder of the embryonic primitive streak, and AX. Arrowhead, node; arrow as in C.

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The significance of Oct-3/4 in AX is less clear. Oct-3/4-positive cells were apparent in this tissue at the EHF stage, and were present until 6-s, after which this endoderm was no longer clearly associated with the allantois. As the AX was found in the extraembryonic region, it may become part of the visceral yolk sac. On the other hand, the AX contained Oct-3/4-positive cells, distinguishing it from all other extraembryonic visceral endoderm, and suggesting that, like the EVE, it will become part of the embryonic hindgut. EVE's ability to shift its position (Thomas and Beddington,1996) provides a fitting paradigm for building the hindgut by AX translocation. Whether the AX becomes part of the hindgut is, however, currently unknown, but loss of obvious physical association between the allantois and AX by 7-s lends support to this possibility.

The Caudal Node: A Stem Cell Reservoir?

By 7.75 dpc, Oct-3/4 was found in neurectoderm and surface ectoderm. In the node, Oct-3/4 was present in all but its ventral anterior aspect. Results of elegant tour-de-force potency studies (Cambray and Wilson,2007) have provided compelling evidence that the anterior portion of the node is not self-renewing; rather it contains a limited reservoir of cells that contributes to the notochord. By contrast, other results have demonstrated that the ventral node contains a self-renewing cell population that acts as a stem cell reservoir for building midline embryonic structures (Beddington,1994; Tam et al.,2004). Moreover, in the chick, fate mapping revealed that Henson's node, analogous to that of the mouse (Beddington,1994), has a complex architecture, various parts of which give rise to multiple cell lineages, including endoderm and neural tube (Selleck and Stern,1991). Together, these results argue that the caudal node, conspicuously Oct-3/4-positive in this study, may be a stem cell reservoir, providing raw materials to expand posterior neurectoderm and the gut during later embryonic stages.

Inconsistencies Between Oct-3/4's Presence and “Stemness” In Vivo

Neural plate/LB anterior epiblast is fated to give rise to anterior neurectoderm (Beddington,1982). Moreover, anterior epiblast loses pluripotency by this stage, failing to contribute to gut endoderm, and preferentially colonizing neurectoderm (Beddington,1982). Thus, despite robust expression of Oct-3/4 (Fig. 2H–J), anterior epiblast is developmentally restricted during early gastrulation. By contrast, distal and posterior epiblast were pluripotent (Beddington,1982) and, in this study, they also exhibited high levels of Oct-3/4 (Fig. 2H–J).

In addition, Oct-3/4 was expressed in both the mid- and proximal allantoic regions until 5-s. Yet, previous heterotopic grafting experiments revealed that the mid-allantoic region of the headfold-stage conceptus colonized only embryonic blood vessels. By contrast, the proximal allantoic region contained cells that colonized derivatives of all three primary germ layers (Downs and Harmann,1997). Together, these data suggest that the presence of Oct-3/4 is not an accurate indicator of pluripotency.

Recently, Oct-3/4's correlation with pluripotency was challenged in several adult settings, ranging from differentiated adult human peripheral blood mononuclear cells that contained Oct-3/4 (Zangrossi et al.,2007), to pluripotent adult stem cells that did not (Ledford,2007). Together with the aforementioned findings, these contradictions raise the serious question of whether Oct-3/4's presence/absence accurately and universally reflects the pluripotent/differentiated state.

In Vitro, Oct-3/4 Expression Accords With Oct-3/4-Positive Tissue Origin

Despite inconsistencies in vivo, Oct-3/4's absence/presence in a variety of stem cell lines in vitro accords with developmental restriction/pluripotency and each cell line's in vivo precursor tissue. For example, trophoblast stem cells, or TSCs, colonize only trophectoderm derivatives and do not express Oct-3/4 (Tanaka et al.,1998). In vivo, Oct-3/4 is down-regulated in trophectoderm at implantation (Nichols et al.,1998; Niwa et al.,2000). Moreover, trophectoderm-derived chorionic ectoderm, a source 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, extraembryonic visceral endoderm, or XEN, stem cells are also developmentally restricted and do not express Oct-3/4 (Kunath et al.,2005). With the exception of the AX (Fig. 3C–G), XVE does not contain Oct-3/4 in vivo (Fig. 3C–G). By contrast, two epiblast-derived stem cell lines, EG cells (Matsui et al.,1992) and EpiSC (Brons et al.,2007; Tesar et al.,2007), both of which are pluripotent, express Oct-3/4 and reflect Oct-3/4's status in vivo in the early post-implantation epiblast (Scholer et al.,1990). Human amniotic stem cells, both those that have been isolated from term amnion (Miki et al.,2005) and those from amniotic fluid (De Coppi et al.,2007), express Oct-3/4 (Figs. 2F,G, 3A–H).

Correlation of Oct-3/4 in epiblast-derived stem cell lines and corresponding tissues in vivo suggests that, whatever the tissue, as long as it expresses Oct-3/4, unique cell lines might be derived from it. Two promising candidates are chorionic mesoderm and the allantois. Between 4- and 8-s, these unite and contribute to the nascent chorio-allantoic placenta (Downs and Gardner,1995), a major site of definitive hematopoiesis (Gekas et al.,2005; Otterbach and Dzierzak,2005). The source of placental hematopoietic cells has not been identified, but both the chorion and allantois exhibit definitive hematopoietic potential (Zeigler et al.,2006). Given the robust levels of Oct-3/4 in chorionic mesoderm and the allantois (Figs. 2G, 3A), their survival in culture (Downs et al.,2001; Zeigler et al.,2006), and the presence of pluripotent cells within the proximal allantois (Downs and Harmann,1997), chorionic mesoderm and the allantois may be ideal tissues for deriving pluripotent, vascular, and/or hematopoietic cell lines.

Do Allantoic Oct-3/4-Positive Cells Contribute to the Germ Line?

A number of studies have suggested that the allantois contains PGCs (Chiquoine,1954; Ozdzenski,1967; Ginsburg et al.,1990). Yet, the PGC founding population, which emerges during early gastrulation, is thought to be small (Lawson and Hage,1994). On that basis, Oct-3/4's widespread abundance in the nascent allantois makes it unlikely that all allantoic Oct-3/4-positive cells are primordial germ cells (Scholer et al.,1990). Nevertheless, given the number of multi-lineage descendents to which Oct-3/4 progenitor populations contribute, the large number of Oct-3/4-expressing cell types, and Oct's localization to germ cells in the genital ridges (Scholer et al.,1990), it is tantalizing to speculate that a subpopulation of allantoic cells contributes to the germ line.

Results of studies here suggest various routes by which allantoic PGCs, if they exist, might become part of the hindgut. First, AX Oct-3/4-positive cells may originate within the allantois and, from there, become part of the hindgut by tissue translocation, as suggested in the “Contribution of Oct-3/4-Positive Cells to Gut Endoderm” section. Although orthotopic grafting experiments failed to identify donor allantoic cells in the hindgut, donor allantoic cells had been introduced into the host's proximal allantois directly through adjacent AX, undoubtedly damaging it and pre-empting assessment of its contribution to definitive endoderm (Downs and Harmann,1997).

Alternatively, allantoic PGCs might enter the blood stream, as allantoic Oct-3/4-positive cells are closely associated with the allantoic blood vessels (Fig. 6F). Although blood-borne delivery of germ cells to the gonads has no precedent in mammals, in mouse testes, time lapse imaging has recently revealed that spermatogonia are located near blood vessels (Yoshida et al.,2007). Moreover, chick PGCs home to the gonadal ridges via the bloodstream (Simon,1960; Fujimoto et al.,1976).

Lastly, allantoic PGCs may become part of the fetal hindgut by direct association between the allantois and hindgut (Fig. 6E). Fate mapping proximal allantoic cells failed to demonstrate contribution to embryonic tissues (Downs and Harmann,1997). Rather, all descendents were found within the allantois, suggesting that, once inside this organ, movement of allantoic cells is from proximal-to-distal. However, grafting into the allantois at a level too far removed from, or distal to, the prospective hindgut to associate with gut endoderm, cannot be ruled out.

Conclusions

Together, these findings present a comprehensive profile of Oct-3/4 during mouse gastrulation. Further, they suggest new roles for Oct-3/4 in many cell types, and call into question Oct-3/4's use as a global “marker” of pluripotency. It is anticipated that this developmental blueprint of Oct-3/4's whereabouts during early murine post-implantation development will guide future investigations into Oct-3/4's manifold and, undoubtedly, complex, roles in differentiation of the epiblast during specific windows of gastrulation.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

All animals were treated in accordance with Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals (Public Law 99-158) as enforced by the University of Wisconsin-Madison. For all experiments, n ≥ 3 specimens for each stage, unless otherwise noted.

Mouse Strains, Animal Husbandry, Dissection, and Staging

The F2 generation of matings between the inbred hybrid strain B6CBAF1/J (Jackson Laboratories) was used (Downs,2007). Flk-1/Oct-3/4 co-localization studies employed a male Kdrtm1Jrt mouse (hereafter referred to as Flk-1lacZ) (Shalaby et al.,1995) as previously described (Inman and Downs,2006b). Animals were maintained under a 12-hr light/dark cycle (lights out 13:00), and estrus selection, dissection, and embryo staging were as previously summarized (Downs and Davies,1993; Downs and Gardner,1995; Downs,2007). Briefly, pregnant females were killed by cervical dislocation, conceptuses were dissected away from decidual tissues, Reichert's membrane and associated trophoblast were reflected, and stages and their equivalent approximate dpc were categorized as follows: early, mid-, and late streak (ES, MS, LS) stages, ∼6.75–7.0 dpc; no allantoic bud (OB, 7.0–7.25 dpc); early and late allantoic bud (EB, LB) stages, ∼7.25–7.5 dpc; early and late headfold (EHF, LHF) stages, ∼7.75–8.0 dpc. Thereafter, staging was by numbers of somite pairs (1–2-s, 8.0–8.25 dpc; 2–4-s, 8.25 dpc; 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 and Immunohistochemistry

Flk-1lacZ conceptuses were fixed in 4% paraformaldehyde (2 hr, 4°C) and stained for X-gal activity as previously described (Downs and Harmann,1997), but for 6 hr only, after which specimens were rinsed in phosphate-buffered saline (PBS, Sigma) and dehydrated in increasing methanols as described below prior to immunostaining.

Immunostaining Bouin's- and 4% paraformaldehyde-fixed and sectioned specimens was as previously described (Inman and Downs,2006a) using primary antibody to Oct-3/4 (N-19, goat polyclonal SC-8628, Santa Cruz Biotechnologies, Santa Cruz, CA) at dilutions of 1/67, 1/100, and 1/250 for 1.5 and 3.0 hr at room temperature, and 18 hr at 4°C. For whole mount immunohistochemistry, all conceptuses were fixed in 4% paraformaldehyde (2 hr, 4°C), and rinsed in phosphate-buffered saline (PBS, Sigma), followed by dehydration in an increasing series of methanols, and indefinite storage at −20°C in absolute methanol. Just prior to immunostaining, the yolk sac was opened up in all specimens ≥ OB stage by gentle tearing with a forceps. For immunostaining, all steps were carried out on a rocking platform at room temperature, with the exception that primary and secondary antibody binding were carried out at 4°C on the rocker. Endogenous hydrogen peroxidase activity was eliminated with 5% hydrogen peroxidase/methanol for 5 hr. This was followed by blocking donkey non-specific antigen sites for 2 hr in PBS containing 5% donkey serum (Chemicon) and 0.1% Triton-X (“PBSST”). Specimens were incubated overnight at 4°C in primary antibody against Oct-3/4 (1/100 dilution in PBSST), and the next day, they were washed 5 times in PBSST for a total period of 5 hr, followed by application of secondary antibody (1/500 dilution in PBSST; biotinylated donkey anti-goat IgG, SC-2042; Santa Cruz Biotechnologies, Santa Cruz, CA) and incubation overnight at 4°C. After 5 hr of rinsing in PBSST, specimens were incubated for 3 hr in ready-to-use ABC reagent (PK-7100, Vector Labs, Burlingame, CA), washed 3 times during 1.5 hr in PBSST, then twice during 20 min in PBT, in which bovine serum albumen (0.02%, Sigma, A-4378) replaced the donkey serum. The antibody complex was visualized with diaminobenzoate chromagen (DAKO Corporation, Carpinteria, CA) for 5 min at room temperature, after which specimens were fixed again in paraformaldehyde at 4°C overnight, oriented transversely, sagitally, or frontally, and embedded, after which they were sectioned at a thickness of 6 μm, dewaxed, counterstained in hematoxylin, coverslipped, and examined in the compound microscope. Control experiments included elimination of antibody, and pre-binding the Oct-3/4 antibody with Oct-3/4 peptide (SC-2042p, Santa Cruz Biotechnologies) for 10 hr at 4°C.

Measuring the Allantoic Oct-3/4 Domain

Sagital and transverse orientations of the allantois were used to measure allantoic length and the length of the Oct-3/4 domain, as briefly described in the text. Sagital measurements were made at the EB and LB stages, using previous criteria that defined the base of the allantois as its site of insertion into the amnion and visceral yolk sac at the headfold stage (Downs and Harmann,1997) (Fig. 3A,B). However, this criterion omitted the Oct-3/4-positive region adjacent to the AX (Fig. 3A,B). Nevertheless, inclusion of this region would not alter the length of the Oct-3/4 domain for these EB and LB stages, which extended throughout the entire allantoic projection (Fig. 4A,B). By the EHF stage, the length of the Oct-3/4 domain began to shorten, from which time on calculations were made in transverse sections. At this EHF and all subsequent stages, the first section of the base of the allantois was distinguished from the boundary with the primitive streak by the precipitous decrease in left-right width of the base of the allantois relative to measurements of the two previous sections (e.g., Fig. 1F–I). Not only did transverse orientations allow detailed views of the intensity of all Oct-3/4 cells at every allantoic level, but they provided a means for appraising its domain with respect to anteroposterior, dorsal-ventral, and left-right coordinates, and accurate counts of AX-containing Oct-3/4 cells. Transverse measurements included the region adjacent to the AX (Fig. 3C). The “straightness” of transverse sections was judged with respect to the AX, whose shape was transitional between squamous (EVE, embryonic visceral endoderm in the embryonic region), and cuboidal (XVE, extraembryonic visceral endoderm in the visceral yolk sac) (Fig. 3C), and generally devoid of large vesicles (not shown). The number of sections containing intensely stained Oct-3/4 cells was determined complete if the next two sections did not contain darkly stained cells, illustrated in a sagital section (Fig. 3C). No effort was made in this study to estimate the extent of tissue shrinkage after histological preparation; thus, all measurements were based on counting the number of uninterrupted serial 6-μm-thickness histological sections.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The author is grateful to Landen Rentmeister for initial technical assistance with Figure 1A, to Dr. Val Wilson, Dr. Kate Storey, and Dr. Claudio Stern for valuable discussions on the node's architecture and stem cell potential, and the National Institutes of Health (RO1HD042706) for support.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  • Beddington RSP. 1982. An autoradiographic analysis of tissue potency in different regions of the embryonic ectoderm during gastrulation in the mouse. J Embryol Exp Morph 69: 265285.
  • Beddington RSP. 1983. The origin of the foetal tissues during gastrulation in the rodent. In: JohnsonMH, editor. Development in Mammals. Amsterdam: Elsevier Science Publishers. p 132.
  • Beddington RSP. 1994. Induction of a second neural axis by the mouse node. Development 120: 613620.
  • Bellomo D, Lander A, Harragan I, Brown NA, 1996. Cell proliferation in mammalian gastrulation: The ventral node and notochord are relatively quiescent. Dev Dyn 205: 471485.
  • Brons IG, Smithers LE, Trotter MW, Rugg-Gunn P, Sun B, Chuva de Sousa Lopes SM, Howlett SK, Clarkson A, Ahrlund-Richter L, Pedersen RA, Vallier L. 2007. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448: 191195.
  • Cambray N, Wilson V. 2007. Two distinct sources for a population of maturing axial progenitors. Development 134: 28292840.
  • Cauffman G, Liebaers I, Van Steirteghem A, Van de Velde H. 2006. POU5F1 isoforms show different expression patterns in human embryonic stem cells and preimplantation embryos. Stem Cells 24: 26852691.
  • Chiquoine AD. 1954. The identification, origin, and migration of the primordial germ cells in the mouse embryo. Anat Rec 118: 135146.
  • De Coppi P, Bartsch G, Siddiqui MM, Xu T, Santos CC, Perin L, Mostoslavsky G, Serre AC, Snyder EY, Yoo JJ, Furth ME, Soker S, Atala A. 2007. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotech 25: 100106.
  • Downs KM. 2007. In vitro culture model for studying vascularization in allantoic explants and allantoic fusion with the chorion. In: SoaresMJ, HuntJS, editors. Methods in Molecular Medicine, Vol. 121: Placenta and trophoblast: methods and protocols, Vol. 1 Totowa, NJ: Humana Press. p 241272.
  • Downs KM, Davies T. 1993. Staging of gastrulation in mouse embryos by morphological landmarks in the dissection microscope. Development 118: 12551266.
  • Downs KM, Gardner RL. 1995. An investigation into early placental ontogeny: allantoic attachment to the chorion is selective and developmentally regulated. Development 121: 407416.
  • Downs KM, Harmann C. 1997. Developmental potency of the murine allantois. Development 124: 27692780.
  • Downs KM, Gifford S, Blahnik M, Gardner RL. 1998. The murine allantois undergoes vasculogenesis that is not accompanied by erythropoiesis. Development 125: 45074521.
  • Downs KM, Temkin R, Gifford S, McHugh J. 2001. Study of the murine allantois by allantoic explants. Dev Biol 233: 347364.
  • Downs KM, McHugh J, Copp AJ, Shtivelman E. 2002. Multiple developmental roles of Ahnak are suggested by localization to sites of placentation and neural plate fusion in the mouse conceptus. Mech Dev 119S: S31S38.
  • Downs KM, Hellman ER, McHugh J, Barrickman K, Inman K. 2004. Investigation into a role for the primitive streak in development of the murine allantois. Development 131: 3755.
  • Drake CJ, Fleming PA. 2000. Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood 95: 16711679.
  • Fujimoto T, Ukeshima A, Kiyofuji R. 1976. The origin, migration and morphology of the primordial germ cells in the chick embryo. Anat Rec 185: 139145.
  • Gekas C, Dieterlen-Lievre F, Orkin SH, Mikkola HKA. 2005. The placenta is a niche for hematopoietic stem cells. Dev Cell 8: 365375.
  • Ginsburg M, Snow MHL, McLaren A. 1990. Primordial germ cells in the mouse embryo during gastrulation. Development 110: 521528.
  • Haar JL, Ackerman GA. 1971. Ultrastructural changes in the mouse yolk sac associated with the initiation of vitelline circulation. Anat Rec 170: 437456.
  • Inman K, Downs KM. 2006a. Localization of Brachyury (T) in embryonic and extraembryonic tissues during mouse gastrulation. Gene Expr Patterns 6: 783793.
  • Inman KE, Downs KM. 2006b. Brachyury is required for elongation and vasculogenesis in the murine allantois. Development 133: 2947-2959.
  • Inman KE, Downs KM. 2007. The murine allantois: emerging paradigms in formation and development of the mammalian umbilical cord and its relation to the fetus. Genesis 45: 237258.
  • Kaufman MH. 1992. The Atlas of Mouse Development. London: Academic Press.
  • Kinder SJ, Tsang TE, Quinlan GA, Hadjantonakis A-K, Nagy A, Tam PPL. 1999. The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo. Development 126: 46914701.
  • Kunath T, Arnaud D, Uy GD, Okamoto I, Chureau C, Yamanaka Y, Heard E, Gardner RL, Avner P, Rossant J. 2005. Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts. Development 132: 16491661.
  • Lavial F, Acloque H, Bertocchini F, MacLeod DJ, Boast S, Bachelard E, Montillet G, Thenot S, Sang HM, Stern CD, Samarut J, Pain B. 2007. The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells. Development 134: 35493563.
  • Lawson KA, Hage W. 1994. Clonal analysis of the origin of primordial germ cells in the mouse. In: ChadwickDJ, MarshJ, editors. Germline development. Chichester: Wiley. p 6884.
  • Lawson KA, Pedersen RA. 1987. Cell fate, morphogenetic movement and population kinetics of embryonic endoderm at the time of germ layer formation in the mouse. Development 101: 627652.
  • Lawson KA, Meneses JJ, Pedersen RA. 1986. Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer. Dev Biol 115: 325339.
  • Lawson KA, Meneses J, Pedersen RA. 1991. Clonal analysis of epiblast fate during germ layer formation in the mouse embryo. Development 113: 891911.
  • Ledford H. 2007. Doubts raised over stem-cell marker. Nature 449: 647.
  • Lewis SL, Tam PP. 2006. Definitive endoderm of the mouse embryo: formation, cell fates, and morphogenetic function. Dev Dyn 235: 23152329.
  • Matsui Y, Zsebo K, Hogan BL. 1992. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70: 841847.
  • Miki T, Lehmann T, Cai H, Stolz DB, Strom SC. 2005. Stem cell characteristics of amniotic epithelial cells. Stem Cells 10: 15491559.
  • Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Schoeler H, Smith A. 1998. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct 4. Cell 95: 379391.
  • Niwa H, Miyazaki J-I, Smith AG. 2000. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24: 372376.
  • Otterbach K, Dzierzak E. 2005. The murine placenta contains hematopoietic stem cells within the vascular labyrinth region. Dev Cell 8: 377387.
  • Ozdzenski W. 1967. Observations on the origin of primordial germ cells in the mouse. Zool Polon 17: 367381.
  • Palmieri SL, Peter W, Hess H, Scholer H. 1994. Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol 166: 259267.
  • Scholer HR, Dressler GR, Balling R, Rohdewohld H, Gruss P. 1990. Oct-4: a germline-specific transcription factor mapping to the mouse t-complex. EMBO J 9: 21852195.
  • Selleck MA, Stern CD. 1991. Fate mapping and cell lineage analysis of Hensen's node in the chick embryo. Development 112: 615626.
  • Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu X-F, Breitman ML, Schuh AC. 1995. Failure of blood-island formation and vasculogenesis in Flk-1 deficient mice. Nature 376: 6266.
  • Simon D. 1960. Contribution a l'étude de la circulation et du transport des gonocytes primaires dans les blastodermes d'Oiseau cultivés in vitro. [Contribution to the study of primordial germ cell transport through the circulation in chick blastoderms cultured in vitro]. Arch Anat Microsc Morph Exp 49: 93176.
  • Stefanovic S, Pucéat M. 2007. Not just a gatekeeper of pluripotency for embryonic stem cell, a cell fate instructor through a gene dosage effect. Cell Cycle 6: 810.
  • Tam PPL, Beddington RSP. 1987. The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis. Development 99: 109126.
  • Tam PP, Beddington RS. 1992. Establishment and organization of germ layers in the gastrulating mouse embryo. In: ChadwickDJ, MarshJ, editors. Postimplantation development in the mouse. Chichester: John Wiley and Sons. p 2741.
  • Tam PP, Khoo P-L, Wong N, Tsang TE, Behringer RR. 2004. Regionalization of cell fates and cell movement in the endoderm of the mosue gastrula and the impact of loss of Lhx1 (Lim1) function. Dev Biol 274: 171187.
  • Tanaka S, Kunath T, Hadjantonakis A-K, Nagy A, Rossant J. 1998. Promotion of trophoblast stem cell proliferation by FGF4. Science 282: 20722075.
  • Tesar PJ, Chenoweth JG, Brook FA, Davies TJ, Evans EP, Mack DL, Gardner RL, McKay RD. 2007. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448: 196199.
  • Thomas PQ, Beddington R. 1996. Anterior primitive endoderm may be responsible for patterning the anterior neural plate in the mouse embryo. Curr Biol 6: 14871496.
  • Uy GD, Downs KM, Gardner RL. 2002. Inhibition of trophoblast stem cell potential in chorionic ectoderm coincides with occlusion of the ectoplacental cavity in the mouse. Development 129: 39133924.
  • Yoshida S, Sukeno M, Nabeshima Y. 2007. A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis. Science 317: 17221726.
  • Zangrossi S, Marabese M, Brogghini M, Giordano R, D'Erasmo M, Montelatici E, Intini D, Neri A, Pesce M, Rebulla P, Lazzari L. 2007. Oct-4 expression in adult human differentiated cells challenges its role as a pure stem cell marker. Stem Cells 25: 16751680.
  • Zeigler BM, Sugiyama D, Chen M, Guo Y, Downs KM, Speck NA. 2006. The allantois and chorion, which are isolated before circulation or chorio-allantoic fusion, have hematopoietic potential. Development 133: 41834192.
  • Zeineddine D, Papadimou E, Chebli K, Gineste M, Liu J, Grey C, Thurig S, Behfar A, Wallace VA, Skerjanc IS, Pucéat M. 2006. Oct-3/4 dose dependently regulates specification of embryonic stem cells toward a cardiac lineage and early heart development. Dev Cell 11: 535546.