The mouse allantois: new insights at the embryonic-extraembryonic interface

Abstract

During the early development of Placentalia, a distinctive projection emerges at the posterior embryonic-extraembryonic interface of the conceptus; its fingerlike shape presages maturation into the placental umbilical cord, whose major role is to shuttle fetal blood to and from the chorion for exchange with the mother during pregnancy. Until recently, the biology of the cord's vital vascular anlage, called the body stalk/allantois in humans and simply the allantois in rodents, has been largely unknown. Here, new insights into the development of the mouse allantois are featured, from its origin and mechanism of arterial patterning through its union with the chorion. Key to generating the allantois and its critical functions are the primitive streak and visceral endoderm, which together are sufficient to create the entire fetal-placental connection. Their newly discovered roles at the embryonic-extraembryonic interface challenge conventional wisdom, including the physical limits of the primitive streak, its function as sole purveyor of mesoderm in the mouse, potency of visceral endoderm, and the putative role of the allantois in the germ line. With this working model of allantois development, understanding a plethora of hitherto poorly understood orphan diseases in humans is now within reach. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Keywords: allantois; arteries; mesoderm; primitive streak; primordial germ cells; visceral endoderm.

Figure 1.

(a) Schematic diagram summarizing development of the allantois according to general morphological descriptions prior to discovery of the streak's posterior extension into the exocoelom. Unless otherwise indicated, all views in this and other Figures are sagittal, showing the conceptus and its allantois through the axial midline. The red ellipse encircles the posterior embryonic–extraembryonic junction, highlighting the assumed posterior limit of the primitive streak within the embryo. Briefly, once formation of the exocoelom (xc) is nearly complete (NP approx. 7.0–7.5), the allantois (al) appears to emanate from the primitive streak. It enlarges, vascularizes, and elongates as a projectile toward the chorion (ch), fusing with it to become the chorio-allantoic placenta. (b) Current view of allantoic development vis-à-vis extension of the primitive streak into the exocoelom and contact with extraembryonic visceral endoderm. Modified and reprinted with permission [3]. Copyright 2020, Wiley. 1. Pre-allantoic bud stage. The primitive streak extends into the exocoelom where it is complexed with extraembryonic visceral endoderm (xve). The arrow in this and the other panels indicates the allantoic/yolk sac junction and at this stage, also, the site of placement and formation of the vessel of confluence resulting from the streak's extension. Also indicated is the extent of the primitive streak's reach in wild-type T⁺/T⁺, heterozygous T^C/T⁺ and homozygous mutant T^C/T^C embryos, with coloured dotted lines corresponding to genotypes. This is further explained in c. 2. Allantoic bud stage. Formation of the allantoic bud begins at the posteriormost extension of the primitive streak; once the bud is in place, streak-associated extraembryonic visceral endoderm is referred to as the ‘AX’. The AX-derived endodermal rod is inferred at this stage [4]. 3. Headfold-early somite stages. The extraembryonic extension of the primitive streak expands into a dense core, the allantoic core domain (ACD), under influence of the AX. The AX's rod-like extension has thread itself into the ACD. Axial visceral endoderm is segmented into three distinct domains: blood island-associated visceral endoderm, distal AX, which has given up its share of mesoderm, and the AX which, via contact with the streak, continues to undergo an EMT. 4. 7–9-somite stages. The ACD has retracted toward the hindgut as the latter formed. The AX has become the hindgut lip. The status of the allantoic rod is not known. (c) Placement of the vessel of confluence according to developmental stage and the status of Brachyury. Red asterisks indicate the stages when the VOC becomes misplaced within the embryo rather than remaining fixed within the extraembryonic region. The downward black arrows indicate the direction of displacement toward the embryo of the allantois-yolk sac junction over time. Reprinted with permission [5]. Copyright 2017, Elsevier. (d) The allantoic bud (al) appears near the posteriormost extension of the primitive streak (black arrow), at some distance from the embryonic–extraembryonic junction (dotted line) and presenting as an appendage issuing from extraembryonic visceral endoderm. Modified and reprinted with permission [6]. Copyright 2009, Elsevier. (e,f) Fate mapping the allantois-associated extraembryonic visceral endoderm (AX). The DiI-labelled whole mount images in (e,f) have not been published but are an accurate representation of those that have been [4]. (e) Whole mount view of a headfold-stage conceptus, and DiI label within the allantois-associated extraembryonic visceral endoderm (black arrow). (f) Ventral whole mount view of the hindgut lip. The labelled AX becomes the hindgut lip (white arrow). Inset: Photobleached histological section showing AX contribution (brown colour) to the full panoply of cells and tissues at the posterior embryonic–extraembryonic interface. Reprinted with permission [4]. Copyright 2017, Elsevier. Abbreviations for this and all following figures: A, anterior; ac, amniotic cavity; ACD, allantoic core domain; al, allantois; al-r, allantoic regenerate; am, amnion; AX, allantois-associated extraembryonic visceral endoderm; bi, blood island; ch, chorion; D, distal; da, dorsal aortae; dAX, distal allantois-associated extraembryonic visceral endoderm; DCM, dorsal cuboidal mesothelium; EHF, early headfold stage; em, embryo; ES, early primitive streak stage; hf, headfold (anatomical); hg, hindgut; hgl, hindgut lip; HF, headfold stages; ips, intraembryonic posterior primitive streak (synonymous with pps); LB, late allantoic bud stage; LHF, late headfold stage; LS, late primitive streak stage; n, node; NP, neural plate stages; ntc, notochord; OB, no allantoic bud stage; P, posterior; pac, proamniotic cavity; pbi, prospective blood island; pps, posterior intraembryonic primitive streak (synonymous with ips); Pr, proximal; ps, primitive streak; pxve, proximal extraembryonic visceral endoderm (i.e. beneath the level of the chorion); r, allantoic rod; s, somite pairs; T, Brachyury; tb, tailbud; ua, umbilical artery; VCM, ventral cuboidal mesothelium; vv, vitelline (yolk sac) vessels; xc, exocoelom; xve, extraembryonic visceral endoderm; y, yolk sac; ys, yolk sac.

Figure 2.

(a) Localization of OCT-3/4 (brown colour) to the AX (arrowheads) and ACD (asterisk; the ACD is described in §9 and further in figure 3a). Modified and reprinted with permission [61]. Copyright 2008, Elsevier. (b) Localization of T (brown colour) to the AX (arrowheads) and ACD (asterisk; §9 and figure 3a). Unpublished transverse immunohistological section through EHF-stage conceptus (approx. E7.75) shows T within the AX and the ventral disposition of the ACD (§9, figure 3a), and is taken from the same embryo reported in figure 1i,j [6]. (c) Localization of Foxa2 (brown colour) to the AX (arrowheads). Modified and reprinted with permission [62]. Copyright 2017, Elsevier. (d) Grafts of the AX, ACD and AX + ACD into host conceptuses. Arrows indicate 1: AX grafts that did not maintain contact with the primitive streak showed formation of blood cells and paucity of mesoderm production. 2: AX + ACD fully recapitulated the embryonic–extraembryonic interface. 3: ACD grafts that lost contact with the AX lost polarity and the dense core. 4: ACD + AX grafts fully recapitulated the embryonic–extraembryonic interface. Modified and reprinted with permission [4]. Copyright 2017, Elsevier. (e–h) Shaping the allantois via Hedgehog signalling. Included is a brief description of allantois vasculogenesis. See text for details. Modified and reprinted with permission [3]. Copyright 2020, Wiley. See abbreviations in figure 1.

Figure 3.

(a) Immunostained section showing localization of T within the base of the allantois of a late headfold stage conceptus (approx. E8.0); this region was since named the allantoic core domain (ACD) (asterisk). Modified and reprinted with permission [63]. Copyright 2006, Elsevier. (b,c) Explanted allantoises on tissue culture plastic, neural plate/late allantoic bud stage (approx. E7.5), in the presence of allantois-associated extraembryonic visceral endoderm (white arrow) (b), and its absence (c). In the absence of the AX, the ACD does not seem to form and the allantois is greatly reduced. Reprinted with permission [6]. Copyright 2009, Elsevier. (d) Schematic diagram, frontal (rear) view, of the posterior side of the conceptus, summarizing the results of DiI labelling the axial midline of the ACD, intraembryonic posterior primitive streak, and the allantoic flanks, both left and right. Reprinted with permission [6]. Inset shows actual result of DiI labelling the axial midline of the ACD, with cells remaining at the site of labelling (white arrowhead), as well as extending further posteriorly (distally) through the axial midline of the allantois. Modified and reprinted with permission [6]. Copyright 2009, Elsevier. (e) Frontal whole mount fluorescence of conceptus whose anteriormost region of the primitive streak, the node (n, white arrow), had been DiI labelled. As with the ACD, cells at the site of labelling remained in place while extending a midline file of cells, here, further anteriorly, to create the notochord (ntc). Modified and reprinted with permission [60]. Copyright 1994, Development. (f) Schematic diagram of late headfold-stage conceptus depicting bifurcation of extraembryonic visceral endoderm (arrow) into the allantois, creating the allantoic rod (r) (arrowhead). Modified and reprinted with permission [64]. Copyright 1974, Company of Biologists (Development). (g) Frontal (rear) view reconstruction of the relationship of the ColIV-positive allantoic rod to the ACD within the allantois. Dotted line represents the lower limit of the allantoic rod; the white asterisk indicates the proximal component of the ACD (figure 4a). Reprinted with permission [4]. Copyright 2017, Elsevier. (h) Ventral whole mount view showing simultaneously DiI-labelled node (n) and ACD (white asterisk) post-culture; DiI remains in place at the site of labelling while sending forth the notochord and allantoic rod further anteriorly and posteriorly, respectively. The gap between the node and notochord (the ‘head process’) has been previously explained [3]. Reprinted and modified with permission [3]. Copyright 2020, Wiley. (i) Frontal (rear view) histological section of an early primitive streak-stage human conceptus (approx. E18.5) showing the pre-umbilical cord consisting of endodermal (‘allantois’ in humans) and mesodermal (‘body stalk’ in humans) components. A discussion of terminology in humans and mouse can be found in [3]. Modified and reprinted from [65], with acknowledgement to the Carnegie Institute of Washington. See abbreviations in figure 1.

Figure 4.

(a) Schematic diagram of headfold-stage allantois divided into four fate-mapped regions and the underlying intraembryonic posterior primitive streak; indications inside each region reveal contributions to the conceptus. Reprinted with permission [3]. Copyright 2020, Wiley. (b) Left panel: Whole mount view of a post-culture donor conceptus whose allantois and ACD were completely removed; the donor ACD was grafted into the conceptus on the right. In the absence of the ACD, the donor's regenerate allantois (al-r) is unable to elongate. Right panel: Whole mount view of post-culture grafted host conceptus whose allantois had been removed and replaced with the donor ACD graft, showing restoration and elongation of the allantoic regenerate (al-r). Modified and reprinted with permission [6]. Copyright 2009, Elsevier. (c) Histological section of chimeric T⁺/T⁺ ↔ T^C/T^C allantois, showing restored elongation (but lack of fusion with the chorion) of mutant T^C/T^C allantois that received a wild-type T⁺/T⁺ ACD. Modified and reprinted with permission [6]. Copyright 2009, Elsevier. (d) Localization of MIXL1 (brown colour; arrowheads within allantois, arrow in AX) to the ACD and AX. Modified and reprinted with permission [81]. Copyright 2014, Elsevier. (e) Frontal (rear) histological section of Mixl1^−/− mutant showing overgrowth of the allantois. The base of the allantois (dotted line) is this author's interpretation, as it is continuous with the amnion. Reprinted with permission [82]. Copyright 2002, Development. (f) Origin of the ACD founder population. Leftmost drawing indicates clonal labelling of cells within Zones I and II (left to right, respectively, outlined in red); rightmost pair of schematic drawings indicates that cells from Zones I and II, respectively, ended up clustered in the base of the allantois after 24 h of whole embryo culture, and thus, appear to indicate the ACD's stem cell population, a portion of which stays in place after labelling (figure 3d,h). Modified and reprinted with permission [26]. Copyright 1991, Development. (g) Activated Caspase-3 localization (arrow) within a unique axial border cell separates the distal AX from the yolk sac blood island visceral endoderm. Asterisk indicates the allantois–yolk sac junction. Reprinted with permission [4]. Copyright 2017, Elsevier. (h) Schematic diagram illustrating the allantois's major blood vessels and mesothelial domains identified by porosity tests, and their possible role in branching vascularization and protection from branching within the allantois. DCM, VCM, dorsal and ventral cuboidal mesothelium, respectively, have limited porosity. See text for details. Asterisk, VOC. Reprinted with permission [66]. Copyright 2011, Wiley. (i) The allantois of this specimen is wedged between the visceral yolk sac and amnion, and thought to be the normal disposition of these tissues in vivo [78], thereby facilitating directed extension to the chorion and the EMT. BMP4 immunostaining (brown colour, arrowheads) is abundant in extraembryonic visceral endoderm, especially allantois-associated extraembryonic visceral endoderm (AX) that is undergoing the EMT, and in distal allantois-associated extraembryonic visceral endoderm (dAX) that is completing and/or has completed the EMT (2-somite stage, approx. E8.25). BMP4 is also observed throughout the ventral component of the allantois that has just undergone shaping via Hedgehog signalling. Inset: A similar immunohistological section taken from the same embryo which shows, at a higher magnification, a freshly delaminated cell (arrow) from the AX, near the BMP4-positive allantois-yolk sac junction. The status of BMP4 in.the allantois and surrounding visceral endoderm was originally described ([93]; Figure 7 of that publication). The antibody used in the latter, obtained from Santa Cruz Biotechnology, was applied following preparation of histological sections. The antibody used to obtain the data in this panel was from Abcam (Ab39973, 0.4 mg ml⁻¹, diluted 1/200) and carried out first in whole embryos followed by embedding in paraffin and sectioning on the microtome, as previously described [61]. Thus, two independent antibodies from different manufacturers and under different conditions produced similar results. The horizontal line in the main panel is the embryonic–extraembryonic junction. K. Downs 2015, unpublished data. See abbreviations in figure 1.

Figure 5.

(a) Summary of current view of the allantois’s architectural features. See text for details. Reprinted with permission [3]. Copyright 2020, Wiley. (b) Modification of the standard fate map of the zygote [84], incorporating definitive endoderm [4,46] and extraembryonic mesoderm (red arrows), both derived from visceral endoderm [4]. Reprinted with permission [4]. Copyright 2017, Elsevier. See abbreviations in figure 1.