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. Author manuscript; available in PMC: 2011 Jan 24.
Published in final edited form as: Dev Dyn. 2009 Dec;238(12):3193–3204. doi: 10.1002/dvdy.22129

Collagen Type IV and Perlecan Exhibit Dynamic Localization in the Allantoic Core Domain, a Putative Stem Cell Niche in the Murine Allantois

Maria M Mikedis 1, Karen M Downs 1,*
PMCID: PMC3025406  NIHMSID: NIHMS262831  PMID: 19924818

Abstract

A body of evidence suggests that the murine allantois contains a stem cell niche, the Allantoic Core Domain (ACD), that may contribute to a variety of allantoic and embryonic cell types. Given that extracellular matrix (ECM) regulates cell fate and function in niches, the allantois was systematically examined for Collagen type IV (ColIV) and Perlecan, both of which are associated with stem cell proliferation and differentiation. Not only was localization of ColIV and Perlecan more widespread during gastrulation than previously reported, but protein localization profiles were particularly robust and dynamic within the allantois and associated visceral endoderm as the ACD formed and matured. We propose that these data provide further evidence that the ACD is a stem cell niche whose activity is synchronized with associated visceral endoderm, possibly via ECM proteins.

Keywords: allantois, Allantoic Core Domain (ACD), amnion, basement membrane, blood islands, chorionic ectoderm, Collagen type IV, gastrulation, heparan sulfate proteoglycan, mesoderm, mouse, niche, Perlecan, primitive streak, Reichert’s membrane, sinuses of Duval, stem cells, vasculogenesis, visceral endoderm, yolk sac

INTRODUCTION

During gestation in eutherian mammals, survival of the fetus depends on the chorioallantoic placenta. This compound organ is generated by fusion between the chorion and allantois, the latter of which elongates from the posterior region of the embryo. Although many mutants bear defects in allantoic elongation (reviewed in Inman and Downs, 2007), the biological context for such gene activity was, until recently, largely unknown.

Investigation into homozygous Brachyury (T) mutants, which produce a foreshortened allantois, revealed the presence of a putative stem cell niche within the proximal core of the allantois (Inman and Downs, 2006a; Downs et al., 2009). Unexpectedly, this Allantoic Core Domain (ACD) was established by an extraembryonic component of the primitive streak (XPS) (Downs et al., 2009). Prior to the appearance of the allantois (No Bud, OB stage, ~7.0 days post-coitum, dpc), the posterior primitive streak extended into the extraembryonic region as a wedge of cells juxtaposed to overlying visceral endoderm. Shortly thereafter (Early Bud, EB stage, ~7.25 dpc), the allantoic bud emerged from the XPS. Then, in collaboration with overlying allantois-associated extraembryonic visceral endoderm (AX), the XPS created the ACD (Late Bud to Early Headfold, LB-EHF stages, ~7.5–7.75 dpc). Consequently, the allantois elongated far enough to fuse with the chorion and form the chorioallantoic placenta (8-somite pairs, -s, ~8.75 dpc).

A variety of observations have provided compelling support that the ACD is a stem cell niche. These observations include the colonization of proximal allantoic cells into all three primary germ layers when placed in ectopic embryonic sites (Downs and Harmann, 1997), ACD rescue of allantoic elongation in embryos bearing foreshortened allantoises (Downs et al., 2009), and persistence of labeled cells within the ACD as other labeled ACD descendants formed a midline file of cells through the allantois (Downs et al., 2009).

In addition, it has been thought for many years that the base of the allantois is the site of primordial germ cell (PGC) formation (Chiquoine, 1954; Ozdzenski, 1967; Ginsburg et al., 1990). PGCs contain nuclear T (Yabuta et al., 2006) and Oct-3/4 (Scholer, 1991), also widespread in the ACD (Inman and Downs, 2006b; Downs et al., 2009). Many of the ACD’s T- and Oct-3/4-positive cells co-express Flk-1 (Downs, 2008; Downs et al., 2009), which identifies angioblasts (Yamaguchi et al., 1993), while others appear to move into the AX (Downs, 2008; Downs et al., 2009) and share a protein profile with definitive endoderm (Wilkinson et al., 1990; Inman and Downs, 2006b; Downs, 2008). Thus, the ACD may contain progenitor cells for a variety of cell types.

As a putative stem cell niche, the ACD should contain a microenvironment that regulates stem cell maintenance, activation, and proliferation through extracellular signaling and cell–cell interactions (Schofield, 1978; reviewed in Watt and Hogan, 2000). Extracellular matrix (ECM) is particularly important in the niche as it not only provides structural support for and anchorage of stem cells, but it also modulates cell signaling (reviewed in Daley et al., 2008). While little is known about the allantoic ECM, absence of T resulted in decreased cell proliferation, fewer Flk-1 angioblasts, and death of the allantoic core (Inman and Downs, 2006a). Intriguingly, defects in ECM structure (Jacobs-Cohen et al., 1983) and cell adhesion (Yanagisawa and Fujimoto, 1977, 1978) are a common theme in T mutations. Furthermore, T mutants exhibit reduced galactosyl-transferase activity (Shur, 1982), suggesting that biosynthesis of collagens (Myllyla et al., 2007) and heparan sulfate proteolgycans (HSPGs) (Bai et al., 2001), as well as cell adhesion to non-fibrous Collagen type IV (ColIV) (Babiarz and Cullen, 1992), are disrupted. Such disruptions may contribute to the ACD defects observed in T mutants.

Two ECM proteins, ColIV and Perlecan, are ideal candidates for a role in the ACD stem cell niche. ColIV promotes the differentiation of Flk-1-positive endothelial cells (Nishikawa et al., 1998; Yamashita et al., 2000) while Perlecan, an HSPG, binds VEGF to regulate Flk-VEGF signaling (Gitay-Goren et al., 1992; Tessler et al., 1994; Lindner et al., 2007). Both Flk-1 and VEGF have been identified in the allantois (Downs et al., 1998, 2001). Furthermore, ColIV and Perlecan are ubiquitous components of the ECM that have been implicated in niche function (Gupta et al., 1998; Ahmad et al., 2007; Kerever et al., 2007).

Although several protein localization studies of the mouse gastrula have reported the presence of ColIV, Perlecan, and other extracellular macromolecules in the ectodermal and endodermal basement membranes, they have failed to detect these same macromolecules in the allantois (Herken and Barrach, 1985; Gersdorff et al., 2005). Intriguingly, one study localized ColIV to the allantois at a single stage (Leivo et al., 1980), but because so little was known about the biology of the allantois at that time, these data could not be interpreted. In light of ColIV and Perlecan’s contributions to endothelial development (Nishikawa et al., 1998; Yamashita et al., 2000; Lindner et al., 2007) and their localization to several stem cell niches where they regulate cell fate (Gupta et al., 1998; Ahmad et al., 2007; Kerever et al., 2007), we used an optimal method of immunohistochemistry based on comparative fixation, sectional, and whole-mount specimen results (Downs, 2008) to localize ColIV and Perlecan to the mouse gastrula (~7.0–8.75 dpc), focusing especially on the posterior region of the embryo and the allantois.

RESULTS

For all tissues in this study, protein localization encompassed the OB-8-s stages (~7.0–8.75 dpc), a period of approximately 42 hr. Within this time period, all tissues were examined at intervals of 2–4 hr in at least three specimens per stage in both sagittal and transverse orientations. All specimens were similarly prepared, from rapid dissection through fixation, immunohistochemistry, and embedding (see Experimental Procedures section). Specimens of diverse stages were immunostained in the same experiment and complete serial sections were made on each conceptus to compare levels of staining intensity within and between stages. Reproducibility of results was based on evaluation of specific gastrulating structures whose timing of appearance was known.

ColIV and Perlecan Localize to the XPS and Base of the Allantois

ColIV localization was previously reported in the allantois (Leivo et al., 1980), but later studies failed to verify these results (Herken and Barrach, 1985; Gersdorff et al., 2005). Since that time, the architecture of the allantois has been shown to be more complex than was previously known. Here, four components of the allantois were examined: the XPS; the XPS-derived ACD; the distal allantoic core; and the allantois’s outer layer of cells, the mesothelium. In addition, we examined the relationship between the ACD and AX.

At the OB stage (~7.0 dpc), both ColIV and Perlecan localized to the XPS (Fig. 1A,D), which extends as a polarized wedge of cells some distance into the exocoelomic cavity adjacent to visceral endoderm (Downs et al., 2009). With the exception of basement membranes that separated distinct tissue types in the mouse gastrula at this time and hereafter (Fig. 1B,E), this concentration of ECM proteins appeared more robust in the XPS than anywhere else. In addition, Perlecan seemed more intense than ColIV, a difference that persisted in the allantois for all stages examined.

Fig. 1.

Fig. 1

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ColIV and Perlecan in the XPS and allantoic bud. Unless otherwise indicated, all sections presented here and in subsequent figures are sagittal with anterior on the left and posterior on the right. ColIV and Perlecan are brown, and all sections were counterstained in hematoxylin (blue color). A, D: OB stage. Line separates intraembryonic primitive streak (ips) from the wedge of XPS extending into exocoloemic cavity (x). Ellipse outlines ColIV (A) or Perlecan (Perl) (D) within the XPS. B, E: LB stage. Arrowheads, ColIV (B) or Perlecan (E) separating ectodermal and mesodermal tissues in the chorion (black), amnion (red), and embryo (black outlined in red). C, F: LB stage, higher magnification of B and E, respectively. Arrow, ColIV (C) or Perlecan (F) in the proximal allantoic bud. Black arrowhead, continuous basement membrane between the bud and allantois-associated extraembryonic visceral endoderm (ax); red arrowhead, loss of this continuous basement membrane in embryonic compartment. al, allantois; ac, amniotic cavity; am, amnion; ch, chorion; xve, extraembryonic visceral endoderm. Scale bar in F = 200 μm (A, C, D, F), 800 μm (B, E).

As the allantoic bud emerged (neural plate/EB and LB stages, ~7.25–7.5 dpc), scattered ColIV and Perlecan signal appeared throughout it (Fig. 1C,F). However, comparison through complete serial sections suggested that the most intense levels of these proteins were found in the proximal allantoic bud (Fig. 1C,F), adjacent to the overlying AX. In addition, ColIV and Perlecan were robust in the basement membrane that separated the allantois from the AX, forming a strikingly continuous and defined border that terminated abruptly within the embryonic compartment (Fig. 1C,F).

By the time the ACD matured (EHF to Late Headfold, LHF stages; ~7.75–8.0 dpc) (Downs et al., 2009), concentrated ColIV and Perlecan persisted in the proximal allantois while extending into the allantoic midline (Fig. 2A,D and data not shown). In particular, ColIV both circumscribed and was present within a small core of cells located in the proximal ventral portion of the EHF stage allantois (Fig. 2A), forming a sub-domain within the T-rich ACD (Fig. 2A, inset). The concentrated Perlecan also overlapped the ACD (Fig. 2D, inset). At this time, both ColIV and Perlecan became discontinuous in the basement membrane lying between the ColIV- and Perlecan-intense core of the allantois and the AX (Fig. 2A,D). The timing of this change in the basement membrane corresponds to when T- and Oct-3/4-positive cells began to appear in the AX (Downs, 2008; Downs et al., 2009). Throughout the more distal reaches of the allantois and outside of the ACD, ColIV and Perlecan were more diffusely localized (Fig. 2A,D).

Fig. 2.

Fig. 2

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ColIV and Perlecan in the allantois (Headfold to 8-s stages). A: EHF stage, ColIV, transverse profile. Inset, EHF stage; T, transverse profile at similar level. Arrowheads, discontinuous ColIV signal along AX. Asterisk outlined in red, ColIV core signal. B: 4-s stage, ColIV. Double asterisks, cluster of cells in the presumptive ACD surrounded by ColIV. Black arrowhead, region along AX with gaps in ColIV signal. Red arrow, ventral mesothelium lined with ColIV. C: 6-s stage, ColIV. Black asterisk, vessel of confluence. Asterisk outlined in red, ACD region with ColIV signal. Arrowhead outlined in red, continuous ColIV signal between allantois and hindgut endoderm. D: EHF stage, Perlecan. Inset, EHF stage; T, similar sagittal profile. Arrowhead, discontinuous Perlecan signal along AX. Asterisk outlined in red, Perlecan core signal. E: 2-s stage, Perlecan, transverse profile. Inset, 2-s stage; T, transverse profile at similar level. Asterisk outlined in red, ACD region with a concentration of Perlecan signal. Black arrowheads, gaps in Perlecan signal along AX. Red arrows, blebs with intense Perlecan signal. F: 6-s stage, Perlecan. Black asterisk, vessel of confluence. Asterisk outlined in red, ACD region with concentrated Perlecan signal. Arrowhead outlined in red, continuous Perlecan signal between allantois and hindgut endoderm. G: 7-s stage, ColIV. Asterisk outlined in red, ACD region with diminished ColIV signal. Black asterisk, omphalomesenteric artery. H: 8-s stage, Perlecan. Askerisk outlined in red, ACD region with diminished Perlecan signal. Black asterisk, vessel of confluence. hg, hindgut; m, allantoic mesothelium. Scale bar in A = 200 μm (A–F, H), 267 μm (G), 453 μm (insets in A, D, E), 2,030 μm (inset in H).

During allantoic elongation (1–6-s; ~8.0–8.5 dpc), intense ColIV and Perlecan persisted in the presumptive ACD, extending from the proximal allantois into the allantoic midline (Fig. 2B,C,E,F), where the central allantoic vessel forms (Inman and Downs, 2006a). Sometimes, ColIV and Perlecan were observed surrounding a cluster of presumptive ACD cells as if separating them from the rest of the allantois (Fig. 2B). Moreover, ColIV and Perlecan localization remained conspicuously discontinuous between the allantois and AX, with gaps potentially large enough for cells to move between the two tissues (Fig. 2B,E). During this time period, Oct-3/4- and T-positive cells continued to be observed in the overlying AX (Downs, 2008; Downs et al., 2009) (Fig. 2E, inset). By 5–6-s, when the hindgut is forming adjacent to the allantois, ColIV and Perlecan once again formed a continuous barrier between them (Fig. 2C,F).

Most of the outer surface of the allantois, so-called “mesothelium.” did not display the continuous signals expected of a basement membrane (Fig. 2A,D). Instead, distal as well as dorsal mesothelium adjacent to the amnion exhibited diffuse ColIV and Perlecan as intense patches within these highly blebbed surfaces (Fig. 2E and data not shown). By contrast, the non-blebbed ventral mesothelial surface adjacent to the ACD was lined with both ColIV and Perlecan (Fig. 2B and data not shown).

By 7- to 8-s, concomitant with loss of the T-defined ACD (Downs et al., 2009), little ColIV and Perlecan signal was observed in the base of the allantois (Fig. 2G,H). At this time, ColIV and Perlecan localized to the basement membrane of the adjacent nascent hindgut endoderm, partitioning it from the allantois. Only allantoic blood vessels, including the vessel of confluence where the umbilical, yolk sac, and fetal vessels join to form a circulatory continuum (Downs et al., 1998), continued to exhibit ColIV and Perlecan; all other parts of the allantois exhibited diminished signals (Fig. 2G,H).

ColIV and Perlecan Localization to Other Extraembryonic Tissues

ColIV and Perlecan also localized to the amnion, yolk sac, chorion, and parietal endoderm.

Amnion

In addition to the basement membrane between amniotic ectoderm and mesoderm (Fig. 3A,E), ColIV and Perlecan were closely associated with amniotic mesoderm, localizing between mesodermal cells (Fig. 3A,E).

Fig. 3.

Fig. 3

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ColIV and Perlecan localize to other extraembryonic tissues. A, E: Amnion. 1-s stage, ColIV (A and inset); EHF stage, Perlecan (E and inset). Arrowhead outlined in red, ColIV- or Perlecan-positive basement membrane between amniotic ectoderm and mesoderm. Arrows, ColIV or Perlecan associated with amniotic mesoderm. B, C, F, G: Visceral yolk sac with blood islands. LHF stage, ColIV (B); 7-s, ColIV (C); LHF stage, Perlecan (F); 8-s stage, Perlecan (G). Arrows, ColIV or Perlecan between mesothelial-endothelial layers; arrowheads, ColIV or Perlecan between XVE and endothelium. Asterisks, blood islands with exemplary ColIV or Perlecan signal. D, H: Chorion. 4-s stage, ColIV (D and inset); 4-s, Perlecan (H and inset). Arrows, ColIV or Perlecan associated with chorionic mesothelium (cm). Arrowhead outlined in red (H), Perlecan-positive basement membrane between chorionic ectoderm (ce) and mesoderm. I, K: Distal XVE and parietal endoderm (pe). LHF stage, ColIV (I); 6-s stage, Perlecan (K). Black arrow, ColIV or Perlecan between distal XVE and chorionic ectoderm; black arrow outlined in red, ColIV-or Perlecan-positive Reichert’s membrane associated with parietal endoderm (pe). Arrowheads (I), ColIV localized to vesicles in distal XVE. J, L: Chorionic ectoderm. 4-s stage, ColIV (J and inset); 6-s stage, Perlecan (L and inset). Arrows, ColIV or Perlecan localized to chorionic ectoderm. Scale bar in A = 80 μm (A, D, E, H, J, L), 133 μm (B, C, F, G, I, K), 453 μm (insets in A, E), 603 μm (insets in D, H), 905 μm (insets in J, L).

Visceral yolk sac

ColIV and Perlecan separated yolk sac endothelium from adjacent mesothelium and extraembryonic visceral endoderm (XVE) (Fig. 3B,C,F,G). ColIV and Perlecan also localized to the basement membrane between chorionic ectoderm and the yolk sac’s distal XVE, which is thought to contribute to the sinuses of Duval in the placenta (Duval, 1891) (Fig. 3I,K). Intriguingly, for all stages examined, ColIV localized to the vesicles within proximal and distal XVE (Fig. 3B,C,I), but Perlecan was not detected in the XVE (Fig 3F,G,K).

From the initiation of yolk sac blood island development (LB stage, ~7.5 dpc) (reviewed in Ferkowicz and Yoder, 2005) through 8-s, ColIV and Perlecan consistently localized between the mesothelial, XVE, and endothelial layers (Fig. 3B,C,F,G). In nascent blood islands, ColIV and Perlecan robustly localized to presumptive hemangioblasts (Fig. 3B,F). With the progression of hemangioblast differentiation, ColIV and Perlecan localization appeared to steadily decrease through 8-s, when the proteins only occasionally localized to yolk sac hematopoietic cells (Fig. 3C,G).

Chorion

ColIV and Perlecan localized to the basement membranes that separated chorionic mesoderm from ectoderm (Fig. 3H and data not shown). In addition, as observed in the amnion, ColIV and Perlecan localized between chorionic mesoderm cells (Fig. 3D,H). Typically, centrally-located chorionic ectoderm contained patches of ColIV and Perlecan (Fig. 3J,L), but during bud stages, occasional localization was also observed outside of this center (data not shown).

Parietal endoderm and Reichert’s membrane

ColIV and Perlecan consistently localized to Reichert’s membrane, associated with parietal endoderm (Fig. 3I,K).

ColIV and Perlecan Localization in Embryonic Tissues

In addition to the ectodermal and endodermal basement membranes, ColIV and Perlecan were detected in all mesodermal tissues and their derivatives within the embryo proper.

Axial mesoderm: the primitive streak, node, and notochord

ColIV and Perlecan were found in the intraembryonic primitive streak (Downs et al., 2009) from the OB to 6-s stages (Fig. 4A,F). By 7- to 8-s, ColIV and Perlecan signals within the primitive streak had diminished (Fig. 4B,G). Where the anterior streak condensed into the node, ColIV and Perlecan separated the node’s dorsal and ventral portions (Fig. 4C,D,H,I). ColIV and Perlecan also separated the notochord and neurectoderm at all stages studied (Fig. 4E,J).

Fig. 4.

Fig. 4

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ColIV and Perlecan in embryonic axial and paraxial mesoderm. A,B, F,G: Primitive streak (ps). 2-s stage, ColIV (A and inset); 8-s stage, ColIV, transverse profile (B); EHF stage, Perlecan (F and inset); 8-s stage, Perlecan (G). Arrowheads (B, G), ColIV or Perlecan staining in primitive streak. C,D, H,I: node (n). 2-s stage, ColIV (C); 6-s stage, ColIV (D); EB stage, Perlecan (H); 6-s stage, Perlecan (I). E, J: Notochord (arrow), transverse profiles with anterior on the top and posterior on the bottom. EHF stage, ColIV (E); LHF stage, Perlecan (J). K,L: Somites (s). 4-s, ColIV (K); 5-s, Perlecan (L). Arrows, somitic basement membrane. Arrowheads, ColIV or Perlecan within somitic lumen. m, mesoderm; ne, neurectoderm; pm, paraxial mesoderm. Scale bar in K = 133 μm (A, E, L), 200 μm (B–D, G–K), 80 μm (F), 1,015 μm (inset in A), 453 μm (inset in F).

Paraxial mesoderm and somites

ColIV and Perlecan localized to paraxial mesoderm (Fig. 4K,L). After somitogenesis, ColIV and Perlecan surrounded each somite while also localizing to the interior lumen of newly formed somites (Fig. 4K,L). However, luminal signals were only occasionally observed in older somites after the formation of the sixth to eighth somite pairs (data not shown).

Lateral plate mesoderm, head mesenchyme, the heart, and blood vessels

The lateral plate mesoderm-derived cardiac field showed ColIV and Perlecan signals throughout cardiogenesis (Fig. 5A–F). Specifically, at the LHF stage (~8.0 dpc), anterior lateral plate mesoderm’s bilateral fields had fused along the ventral midline to form the cardiac field, where ColIV and Perlecan were weakly localized to the mesoderm (Fig. 5D and data not shown). As the heart field developed into a tube, ColIV and Perlecan were found between myocardial layers and along the endocardium of the developing heart from 1- to 8-s (Fig. 5A–C,E,F).

Fig. 5.

Fig. 5

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ColIV and Perlecan in the heart and embryonic vasculature. A–F: Heart (ht). 2-s stage, ColIV (A); 4-s stage, ColIV (B); 7-s stage, ColIV (C); LHF stage, Perlecan (D); 4-s stage, Perlecan (E); 8-s stage, Perlecan, transverse profile (F). Arrows (A, B, D, E), ColIV or Perlecan in head mesenchyme. Single asterisk (C,F), endocardium; double asterisks (C,F), myocardium. G–J: Anterior (G,I) and posterior (H,J) dorsal aorta, transverse profiles with posterior on the top and anterior on the bottom (G,I) or anterior on the top and posterior on the bottom (H,J). 8-s stage, ColIV (G); 8-s stage, ColIV (H); 8-s stage, Perlecan (I); 8-s stage, Perlecan (J). Asterisks, embryonic vessels. hf, heart field. Scale bar in A = 200 μm (A–C, E–J); 80 μm (D).

ColIV and Perlecan also localized to the endothelium of the embryonic vasculature. By 8-s, endothelial cells had shaped the dorsal aorta along the foregut and hindgut. Caudal to the hindgut, the dorsal aorta had fused with the allantoic and vitelline vasculatures. The primary head veins had also formed along the neurectoderm (Kaufman, 1992). ColIV and Perlecan, localized to endothelial cells and surrounded all embryonic vessels by the 8-s stage (Fig. 5G–J). However, ColIV and Perlecan signals at the anterior vessels were weaker than those at posterior vessels (compare Fig. 5G,I to 5H,J). The omphalomesenteric artery adjacent to the hindgut (Fig. 2G) and the vessel of confluence (Fig. 2H) were also ColIV- and Perlecan-positive.

Finally, head mesenchyme exhibited weak ColIV and Perlecan signals until the 7- to 8-s stages (compare Fig. 5A,B,D,E with 5G,I).

DISCUSSION

Summary of ColIV and Perlecan in the Mouse Gastrula

Our results corroborate those of an earlier study reporting ColIV in a variety of tissues at embryonic days 7 and 8 (Leivo et al., 1980). In particular, ColIV localized to visceral endoderm, embryonic mesoderm, and the allantois (Leivo et al., 1980). Our systematic study, which examined fifteen morphological stages of the mouse gastrula, expanded those results, and revealed extensive localization of ColIV and Perlecan to all types of embryonic mesoderm (axial, paraxial, and lateral plate), extraembryonic mesoderm (amniotic, chorionic, yolk sac, and allantoic), and, additionally, the chorionic ectoderm throughout gastrulation.

Do ColIV and Perlecan Contribute to Mesoderm Formation?

Embryonic mesoderm is thought to be derived from primitive ectoderm, or epiblast, that passes through the embryonic primitive streak and undergoes an epithelial-to-mesenchymal transition (EMT) (Beddington, 1983). The ECM contributes to this process by facilitating changes in cell adhesion (Burdsal et al., 1993) and assisting in the migration of mesodermal cells (Klinowska et al., 1994). In this study, ColIV and Perlecan localized to the primitive streak from the OB to 6-s stages, after which point their signals greatly diminished. Although the time period during which epiblast is converted to mesoderm is not known, these observations suggest that ColIV and Perlecan are involved in early mesoderm formation and, thus, the early phases of gastrulation. Indeed, ColIV and Perlecan are required for gastrulation in sea urchin (Wessel and McClay, 1987) and chick (Soulintzi and Zagris, 2007), respectively. While ColIV’s role in gastrulation has not been studied in vertebrae, murine and zebrafish gastrulation do not require Perlecan (Arikava-Hirasawa et al., 1999; Costell et al., 1999; Zoeller et al., 2008). However, if Perlecan’s role in the streak is heparan sulfate–dependent, other HSPGs could provide the functional redundancy needed for murine or zebrafish gastrulation to occur in the absence of Perlecan.

Although conventional wisdom maintains that all mesoderm, both embryonic and extraembryonic, is derived from the embryonic primitive streak (Beddington, 1983), lineage tracing experiments have shown limited contribution from the intraembryonic primitive streak to the allantois (Lawson et al., 1991). Therefore, an additional source of mesoderm, perhaps devoted to producing extraembryonic mesoderm, may exist in the murine conceptus. For example, Bonnevie previously proposed that murine extraembryonic mesoderm delaminates from extraembryonic ectoderm (Bonnevie, 1950; reviewed in Downs, 2009). Surprisingly, we detected ColIV and Perlecan in chorionic ectoderm, an extraembryonic tissue. By contrast, embryonic ectoderm did not contain ColIV or Perlecan signals, and other non-mesodermal signals were associated with basement membranes or, in the case of ColIV, visceral endodermal vesicles. Given that extraembryonic and chorionic ectoderm contain T (Rivera-Perez and Magnuson, 2005; Inman and Downs, 2006b), it is tantalizing to speculate that chorionic ectoderm, like the primitive streak, undergoes an EMT and produces extraembryonic mesoderm.

ColIV and Perlecan in the Posterior Embryonic Region and Allantois

The network-forming macromolecule ColIV regulates stem cell proliferation and differentiation by interacting with growth factors (Balduino et al., 2005; Wang et al., 2008). Perlecan binds numerous growth factors and consequently protects them from degradation, regulates their accessibility, and modulates signaling effects on neighboring cells (reviewed in Iozzo, 2005). For all stages examined here, both ColIV and Perlecan robustly localized to the little-studied posterior region of the conceptus in a dynamic manner typical of development, when ECM undergoes constant remodeling (reviewed in Daley et al., 2008). While ColIV and Perlecan were widely distributed in the conceptus, their dynamic localization patterns within the posterior region and allantois, summarized below and in Figure 6, supported the latter’s biological activities, which are just coming to light.

Fig. 6.

Fig. 6

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Summary of ColIV and Perlecan localization during allantoic ontogeny. A: OB stage. The ColIV- and Perlecan-positive core (yellow) appears in the posterior extraembryonic primitive streak (xps; pink). ColIV and Perlecan form a distinct basement membrane (yellow) between the allantois and extraembryonic visceral endoderm (hatched). B, C: EB and LB stages. The ColIV-and Perlecan-positive core and basement membrane persist as the allantoic bud appears (B) and elongates (C). D: Headfold (HF) stages. With ACD (pale orange) formation, the ColIV- and Perlecan-positive core expands to form a subregion within the ACD. ColIV and Perlecan signals between the allantois and AX (dark green) become discontinuous where the ColIV and Perlecan core meet the AX (dotted yellow and black). This coincides with the appearance of T- and Oct-3/4-positive nuclei (pale orange circles) in the AX. E: 1- to 4-s stages. The ColIV- and Perlecan-positive core in the ACD persists while the discontinuous signals expand along the entire AX. T-and Oct-3/4-positive nuclei persist in the AX. F: 5- to 6-s stages. The ColIV- and Perlecan-positive core persists in the base of the allantois as the ColIV- and Perlecan-positive basement membrane between the allantois and AX regains continuity as the hindgut forms. Embryonic endoderm (light green); ips, intraembryonic primitive streak (red).

ColIV and Perlecan in the XPS May Contribute to the Establishment/Maintenance of the ACD

Just before the appearance of the allantoic bud, ColIV and Perlecan localized to what appeared to be the XPS’s leading edge (Fig. 6A). Although the XPS has only recently been identified (Downs et al., 2009), the alkaline phosphatase-positive cells in this hitherto undefined region of the conceptus are thought to be the PGCs (reviewed in Chuva de Sousa Lopes and Roelen, 2008). PGC specification, though poorly understood, involves Bone Morphogenetic Protein (BMP) and Transforming Growth Factor β (TGFβ) (reviewed in Chuva de Sousa Lopes and Roelen, 2008). Both growth factors have been identified in the allantois (Dickson et al., 1993; Lawson et al., 1999; Downs et al., 2004). Thus, as ColIV and Perlecan modulate BMP (Wang et al., 2008) and TGFβ signaling (Butzow et al., 1993; Lyon et al., 1997; Chen et al., 2007), respectively, ColIV and Perlecan in the XPS may provide the microenvironment required for PGC specification.

The cues that induce formation of the allantoic bud and the ACD from the XPS are not known, but recent data suggest that the AX plays a major role (Downs et al., 2009). During this time period, ColIV and Perlecan formed a distinctly continuous ECM structure between the XPS/allantoic bud and the AX (Fig. 6B,C). This prominent AX-associated basement membrane may facilitate induction of the ACD by sequestering growth factors and modulating their effects on the XPS.

After ACD formation, the ColIV and Perlecan allantoic core was adjacent to the proximal, but not distal, AX before extending into the allantoic midline (Fig. 6D–F). The significance of this subregion is not known, but it overlaps the ACD. Here, ColIV and Perlecan may promote the proliferation of stem cells (Park et al., 2003; Balduino et al., 2005; Kerever et al., 2007). Regulation of proliferation may involve Perlecan’s heparan sulfate chains (Friedrich et al., 1999; Tapanadechopone et al., 1999) enhancing the signaling of Fibroblast Growth Factor (FGF)-8 (Loo and Salmivirta, 2002), which transiently localizes to the base of the allantois (Crossley and Martin, 1995). Disappearance of ColIV and Perlecan after 6-s coincides with that of the ACD (Downs et al., 2009). Together, these data suggest that ColIV and Perlecan play important roles in the ACD and support existing evidence that this region is a stem cell niche.

ColIV and Perlecan in Vasculogenesis/Hematopoiesis of the Allantois

ColIV and Perlecan extended from the ACD core through the allantoic midline to the distal allantois (Fig. 6E,F), possibly guiding and/or facilitating differentiation into the primary Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1)/Flk-1-defined umbilical vasculature (Inman and Downs, 2006a). As ColIV promotes differentiation of murine embryonic stem cells into Flk-1-positive endothelial cells (Nishikawa et al., 1998; Yamashita et al., 2000), allantoic ColIV may facilitate differentiation of ACD cells into the umbilical vasculature. Although Flk-1 is expressed in allantoic blood vessels as late as 10-s (Downs et al., 1998), ColIV is significantly down-regulated in the allantois by 7- to 8-s, perhaps indicating the end of angioblast conversion into endothelium.

Similarly, Perlecan may also play a role in allantoic vasculogenesis. Perlecan was originally identified as an angiogenic factor (Aviezer et al., 1994) that localized to the heart, pericardium, and embryonic blood vessels during organogenesis in the mouse embryo (Handler et al., 1997; Jiang et al., 2004). Localization of Perlecan to the heart and blood vessels throughout the conceptus during this study accords with a role in vascularization. Perlecan, whose heparan sulfate chains interact with PECAM-1 (Coombe et al., 2008), was found near the PECAM-1 central allantoic vessel, just beneath the outer ventral mesothelial surface, and in the vessel of confluence. Perhaps Perlecan facilitates vascular patterning by modulating the signaling of VEGF (Gitay-Goren et al., 1992; Tessler et al., 1994; Lindner et al., 2007), which was anecdotally observed in this sub-region of the mesothelium (Miquerol et al., 1999). Intriguingly, no vasculogenic defects have been reported in Perlecan-null mutants (Arikava-Hirasawa et al., 1999; Costell et al., 1999; Zoeller et al., 2008), but if Perlecan’s heparan sulfates are responsible for its vasculogenic properties, then any other HSPGs that may localize to the allantois could compensate for the loss of Perlecan during vasculogenesis.

The allantois contains definitive hematopoietic potential (Zeigler et al., 2006), but the location and timing of the cells bearing this potential have not been identified. ColIV and Perlecan may contribute to hematopoiesis in the allantois, as ColIV promotes hematopoietic differentiation (Nishikawa et al., 1998; Balduino et al., 2005), while HSPGs regulate hematopoietic stem cell proliferation through FGF signaling (Aviezer et al., 1994; Gupta et al., 1998; Knox et al., 2002). The hematopoietic stem cells may originate within the ACD and/or within superficial allantoic blebs, which, like the ACD, exhibited intense ColIV and Perlecan. Although nothing is known about the function of allantoic blebbing, one possibility is that blebs are microniches critical for hematopoiesis, especially as BMP-4, a hematopoietic factor (Baron, 2003), interacts with ColIV (Sadlon et al., 2004; Wang et al., 2008) and is highly expressed in the outer surface of the allantois (Lawson et al., 1999; Downs et al., 2004).

Allantoic ColIV and Perlecan in PGC Migration to the Hindgut

As the ACD was forming, the hitherto continuous ColIV and Perlecan structure lying between it and the AX became discontinuous (Fig. 6D,E). During development, ECM degradation promotes cell migration by eliminating physical barriers, modifying the ECM’s effects on extracellular signaling, and removing cell adhesion molecules (reviewed in Fata et al., 2004). In the allantois, breakdown of the intervening basement membrane may promote migration of T- and Oct-3/4-positive ACD cells into the AX (Downs, 2008; Downs et al., 2009), where these cells may contribute to definitive hindgut endoderm and/or the PGC population (Downs, 2008). Later, when continuous ColIV and Perlecan signals reappear along the AX during hindgut formation at 5- to 6-s (Fig. 6F), cell movement from the allantois into the AX may be hindered. However, whether the AX, like other visceral endoderm, becomes incorporated into definitive endoderm (Kwon et al., 2008) is not yet known.

PGC movement from the base of the allantois into the hindgut endoderm requires Steel factor, which surrounds presumptive PGCs within the base of the allantois (Gu et al., 2009). While not required for PGC directionality (Gu et al., 2009), Steel factor promotes cell adhesion to fibronectin (Kinashi and Springer, 1994), which localizes to the allantois (Yang et al., 1995). Whether Steel factor interacts with Perlecan is not known; however, Perlecan may indirectly interact with Steel factor by binding to fibronectin (Hopf et al., 1999). ColIV may also influence PGC movement into endoderm, as PGCs adhere to ColIV during their migration through the hindgut (Garcia-Castro et al., 1997).

Conclusions

Until the ACD is carefully fate and potency mapped, and the associated ECM manipulated, our interpretation of the role of ECM proteins in this unique posterior region is necessarily speculative. Results here represent just the beginning in the long road ahead to defining the molecular properties of the ACD. However, placing these results within the context of the emerging biology of the allantois creates a solid framework upon which to build and challenge the significance of these observations.

EXPERIMENTAL PROCEDURES

Mouse Strains, Animal Husbandry, Dissection, and Staging

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. Animals were maintained under a 12-hr light/dark cycle (lights out at 13:00 or 21:00), and estrus selection and mating were as previously summarized (Downs, 2006). Matings between the inbred hybrid strain B6CBAF1/J (The Jackson Laboratory, Bar Harbor, ME) provided the F2 generation for the ColIV and Perlecan localization studies (Downs, 2006). Pregnant females were sacrificed by cervical dislocation, conceptuses were dissected from decidua, Reichert’s membrane was reflected, and embryos were staged (Downs and Davies, 1993) as follows: no bud (OB; ~7.0 dpc); early and late bud (EB, LB; ~7.25–7.5 dpc); early and late headfold (EHF, LHF; ~7.75–8.0 dpc); 1–6-s (~8.0–8.5 dpc), and 7–8-s (~8.5–8.75).

Immunohistochemistry

Previous studies demonstrated that the strength of the primary antibody signal varies among fixatives used to prepare specimens for immunostaining (Downs et al., 2002; Inman and Downs, 2006b; Downs, 2008). Previous ColIV localization studies used 4% PFA-fixed or tannic acid fixed sections for immunohistochemical analysis or 3.5% PFA-fixed sections for immunofluorescent analysis (Leivo et al., 1980; Herken and Barrach, 1985; Gersdorff et al., 2005). Perlecan was previously localized in 4% PFA-fixed sections (Gersdorff et al., 2005). In this study, we localized ColIV and Perlecan using whole mount immunostaining, which was carried out as previously described (Downs, 2008). Throughout this study, ≥3 specimens/stage were used. ColIV staining used anti-Collagen type IV (ab19808; Abcam; Cambridge, MA; 1/500 dilution) and biotinylated donkey anti-rabbit IgG secondary antibody (sc-2089; Santa Cruz Biotechnologies; 1/500 dilution). Perlecan staining used anti-Perlecan (rat anti-mouse anti-heperan sulfate proteogylcan Mab1948; Chemicon; Temecula, CA; 1/100 dilution) and biotinylated goat anti-rat IgG secondary antibody (sc-2041; Santa Cruz Biotechnologies; Santa Cruz, CA; 1/500 dilution). The antibody complex was visualized with diaminobenzoate chromagen (DAB; DAKO Corporation, Carpinteria, CA) at room temperature for 5 min for ColIV and 7.5 min for Perlecan, after which specimens were fixed in 4% paraformaldehyde (PFA) overnight at 4°C. Then, after standard dehydration and clearing, specimens were embedded in wax for transverse or sagittal orientations, sectioned at a thickness of 6 micrometers (microns, μm), dewaxed, counterstained with hematoxylin, coverslipped, and analyzed. Because the thickness of these sections oftentimes created ambiguity concerning the exact intra- or extracellular whereabouts of ColIV and Perlecan signals, we have refrained from reporting such designations except where unambiguous. Negative controls in which the primary antibody was eliminated at 4-s stages (ColIV) and 5-s stages (Perlecan) (N = 3 specimens), when staining signals were most abundant, verified anti- body specificity (Fig. 7).

Fig. 7.

Fig. 7

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ColIV and Perlecan controls. A,C: Staining with antibody (+Ab). 4-s stage, ColIV (A); 5-s stage, Perlecan (C). B,D: Minus antibody controls (−Ab). 4-s stage, ColIV (C); 5-s stage, Perlecan (D). gc, trophoblast giant cells; hf, headfolds. Scale bar in A = 800 μm (A–B, D), 940 μm (C).

Acknowledgments

The authors are grateful to Dr. Kimberly Inman and Christopher Smith for preliminary data on Perlecan in the allantois and to Dexter Jin for assistance on Figure 6. This study was supported by grants from the March of Dimes and National Institutes of Child Health and Development (K.M.D.).

Grant sponsors: March of Dimes; National Institutes of Child Health and Development.

ABBREVIATIONS

ACD

Allantoic Core Domain

AX

allantois-associated extraembryonic visceral endoderm

BMP

bone morphogenetic protein

ColIV

Collagen type IV

dpc

days post coitum

EB

early bud

ECM

extracellular matrix

EHF

early headfold

EMT

epithelial-to-mesenchymal transition

FGF

fibroblast growth factor

HSPG

heparan sulfate proteoglycan

LB

late bud

LHF

late headfold

OB

no bud

PECAM1

platelet endothelial cell adhesion molecule 1

PGC

primordial germ cell

T

brachyury

TGFβ

transforming growth factor β

VEGF

vascular endothelial growth factor

XPS

extraembryonic primitive streak

XVE

extraembryonic visceral endoderm

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