Visceral endoderm and the primitive streak interact to build the fetal-placental interface of the mouse gastrula.

Hypoblast/visceral endoderm assists in amniote nutrition, axial positioning and formation of the gut. Here, we provide evidence, currently limited to humans and non-human primates, that hypoblast is a purveyor of extraembryonic mesoderm in the mouse gastrula. Fate mapping a unique segment of axial extraembryonic visceral endoderm associated with the allantoic component of the primitive streak, and referred to as the "AX", revealed that visceral endoderm supplies the placentae with extraembryonic mesoderm. Exfoliation of the AX was dependent upon contact with the primitive streak, which modulated Hedgehog signaling. Resolution of the AX's epithelial-to-mesenchymal transition (EMT) by Hedgehog shaped the allantois into its characteristic projectile and individualized placental arterial vessels. A unique border cell separated the delaminating AX from the yolk sac blood islands which, situated beyond the limit of the streak, were not formed by an EMT. Over time, the AX became the hindgut lip, which contributed extensively to the posterior interface, including both embryonic and extraembryonic tissues. The AX, in turn, imparted antero-posterior (A-P) polarity on the primitive streak and promoted its elongation and differentiation into definitive endoderm. Results of heterotopic grafting supported mutually interactive functions of the AX and primitive streak, showing that together, they self-organized into a complete version of the fetal-placental interface, forming an elongated structure that exhibited A-P polarity and was composed of the allantois, an AX-derived rod-like axial extension reminiscent of the embryonic notochord, the placental arterial vasculature and visceral endoderm/hindgut.

STELLA collaborates in distinct mesendodermal cell subpopulations at the fetal-placental interface in the mouse gastrula.

The allantois-derived umbilical component of the chorio-allantoic placenta shuttles fetal blood to and from the chorion, thereby ensuring fetal-maternal exchange. The progenitor populations that establish and supply the fetal-umbilical interface lie, in part, within the base of the allantois, where the germ line is claimed to segregate from the soma. Results of recent studies in the mouse have reported that STELLA (DPPA-3, PGC7) co-localizes with PRDM1 (BLIMP1), the bimolecular signature of putative primordial germ cells (PGCs) throughout the fetal-placental interface. Thus, if PGCs form extragonadally within the posterior region of the mammal, they cannot be distinguished from the soma on the basis of these proteins. We used immunohistochemistry, immunofluorescence, and confocal microscopy of the mouse gastrula to co-localize STELLA with a variety of gene products, including pluripotency factor OCT-3/4, mesendoderm-associated T and MIXl1, mesendoderm- and endoderm-associated FOXa2 and hematopoietic factor Runx1. While a subpopulation of cells localizing OCT-3/4 was always found independently of STELLA, STELLA always co-localized with OCT-3/4. Despite previous reports that T is involved in specification of the germ line, co-localization of STELLA and T was detected only in a small subset of cells in the base of the allantois. Slightly later in the hindgut lip, STELLA+/(OCT-3/4+) co-localized with FOXa2, as well as with RUNX1, indicative of definitive endoderm and hemangioblasts, respectively. STELLA was never found with MIXl1. On the basis of these and previous results, we conclude that STELLA identifies at least five distinct cell subpopulations within the allantois and hindgut, where they may be involved in mesendodermal differentiation and hematopoiesis at the posterior embryonic-extraembryonic interface. These data provide a new point of departure for understanding STELLA's potential roles in building the fetal-placental connection.

Brachyury drives formation of a distinct vascular branchpoint critical for fetal-placental arterial union in the mouse gastrula.

How the fetal-placental arterial connection is made and positioned relative to the embryonic body axis, thereby ensuring efficient and directed blood flow to and from the mother during gestation, is not known. Here we use a combination of genetics, timed pharmacological inhibition in living mouse embryos, and three-dimensional modeling to link two novel architectural features that, at present, have no status in embryological atlases. The allantoic core domain (ACD) is the extraembryonic extension of the primitive streak into the allantois, or pre-umbilical tissue; the vessel of confluence (VOC), situated adjacent to the ACD, is an extraembryonic vessel that marks the site of fetal-placental arterial union. We show that genesis of the fetal-placental connection involves the ACD and VOC in a series of steps, each one dependent upon the last. In the first, Brachyury (T) ensures adequate extension of the primitive streak into the allantois, which in turn designates the allantoic-yolk sac junction. Next, the streak-derived ACD organizes allantoic angioblasts to the axial junction; upon signaling from Fibroblast Growth Factor Receptor-1 (FGFR1), these endothelialize and branch, forming a sprouting VOC that unites the umbilical and omphalomesenteric arteries with the fetal dorsal aortae. Arterial union is followed by the appearance of the medial umbilical roots within the VOC, which in turn designate the correct axial placement of the lateral umbilical roots/common iliac arteries. In addition, we show that the ACD and VOC are conserved across Placentalia, including humans, underscoring their fundamental importance in mammalian biology. We conclude that T is required for correct axial positioning of the VOC via the primitive streak/ACD, while FGFR1, through its role in endothelialization and branching, further patterns it. Together, these genetic, molecular and structural elements safeguard the fetus against adverse outcomes that can result from vascular mispatterning of the fetal-placental arterial connection.

Extragonadal primordial germ cells or placental progenitor cells?

Primordial germ cells (PGCs), the precursors of the gametes, are now claimed to segregate within the extra-embryonic tissues of three species of placental mammals. In this brief Commentary, I raise the question of whether the so-called PGCs are not PGCs at all, but rather, progenitor cells that build the fetal-placental interface in Placentalia.

PRDM1/BLIMP1 is widely distributed to the nascent fetal-placental interface in the mouse gastrula.

BACKGROUND: PRDM1 is a transcriptional repressor that contributes to primordial germ cell (PGC) development. During early gastrulation, epiblast-derived PRDM1 is thought to be restricted to a lineage-segregated germ line in the allantois. However, given recent findings that PGCs overlap an allantoic progenitor pool that contributes widely to the fetal-umbilical interface, posterior PRDM1 may also contribute to soma. RESULTS: Within the posterior mouse gastrula (early streak, 12-s stages, embryonic days ∼6.75-9.0), PRDM1 localized to all tissues containing putative PGCs; however, PRDM1 was also found in all three primary germ layers, their derivatives, and two presumptive growth centers, the allantoic core domain and ventral ectodermal ridge. While PRDM1 and STELLA colocalized predominantly within the hindgut, where putative PGCs reside, other colocalizing cells were found in non-PGC sites. Additional PRDM1 and STELLA cells were found independent of each other throughout the posterior region, including the hindgut. The Prdm1-Cre-driven reporter supported PRDM1 localization in the majority of sites; however, some Prdm1 descendants were found in sites independent of PRDM1 protein, including allantoic mesothelium and hindgut endoderm. CONCLUSIONS: Posterior PRDM1 contributes more broadly to the developing fetal-maternal connection than previously recognized, and PRDM1 and STELLA, while overlapping in putative PGCs, also co-localize in several other tissues. Developmental Dynamics 246:50-71, 2017. © 2016 Wiley Periodicals, Inc.

Irx4 Marks a Multipotent, Ventricular-Specific Progenitor Cell.

While much progress has been made in the resolution of the cellular hierarchy underlying cardiogenesis, our understanding of chamber-specific myocardium differentiation remains incomplete. To better understand ventricular myocardium differentiation, we targeted the ventricle-specific gene, Irx4, in mouse embryonic stem cells to generate a reporter cell line. Using an antibiotic-selection approach, we purified Irx4+ cells in vitro from differentiating embryoid bodies. The isolated Irx4+ cells proved to be highly proliferative and presented Cxcr4, Pdgfr-alpha, Flk1, and Flt1 on the cell surface. Single Irx4+ ventricular progenitor cells (VPCs) exhibited cardiovascular potency, generating endothelial cells, smooth muscle cells, and ventricular myocytes in vitro. The ventricular specificity of the Irx4+ population was further demonstrated in vivo as VPCs injected into the cardiac crescent subsequently produced Mlc2v+ myocytes that exclusively contributed to the nascent ventricle at E9.5. These findings support the existence of a newly identified ventricular myocardial progenitor. This is the first report of a multipotent cardiac progenitor that contributes progeny specific to the ventricular myocardium. Stem Cells 2016;34:2875-2888.

The hypoblast (visceral endoderm): an evo-devo perspective.

When amniotes appeared during evolution, embryos freed themselves from intracellular nutrition; development slowed, the mid-blastula transition was lost and maternal components became less important for polarity. Extra-embryonic tissues emerged to provide nutrition and other innovations. One such tissue, the hypoblast (visceral endoderm in mouse), acquired a role in fixing the body plan: it controls epiblast cell movements leading to primitive streak formation, generating bilateral symmetry. It also transiently induces expression of pre-neural markers in the epiblast, which also contributes to delay streak formation. After gastrulation, the hypoblast might protect prospective forebrain cells from caudalizing signals. These functions separate mesendodermal and neuroectodermal domains by protecting cells against being caught up in the movements of gastrulation.

Irx4 identifies a chamber-specific cell population that contributes to ventricular myocardium development.

BACKGROUND: The ventricular myocardium is the most prominent layer of the heart, and the most important for mediating cardiac physiology. Although the ventricular myocardium is critical for heart function, the cellular hierarchy responsible for ventricle-specific myocardium development remains unresolved. RESULTS: To determine the pattern and time course of ventricular myocardium development, we investigated IRX4 protein expression, which has not been previously reported. We identified IRX4+ cells in the cardiac crescent, and these cells were positive for markers of the first or second heart fields. From the onset of chamber formation, IRX4+ cells were restricted to the ventricular myocardium. This expression pattern persisted into adulthood. Of interest, we observed that IRX4 exhibits developmentally regulated dynamic intracellular localization. Throughout prenatal cardiogenesis, and up to postnatal day 4, IRX4 was detected in the cytoplasm of ventricular myocytes. However, between postnatal days 5­6, IRX4 translocated to the nucleus of ventricular myocytes. CONCLUSIONS: Given the ventricle-specific expression of Irx4 in later stages of heart development, we hypothesize that IRX4+ cells in the cardiac crescent represent the earliest cell population in the cellular hierarchy underlying ventricular myocardium development.

STELLA-positive subregions of the primitive streak contribute to posterior tissues of the mouse gastrula.

The developmental relationship between the posterior embryonic and extraembryonic regions of the mammalian gastrula is poorly understood. Although many different cell types are deployed within this region, only the primordial germ cells (PGCs) have been closely studied. Recent evidence has suggested that the allantois, within which the PGCs temporarily take up residence, contains a pool of cells, called the Allantoic Core Domain (ACD), critical for allantoic elongation to the chorion. Here, we have asked whether the STELLA-positive cells found within this region, thought to be specified PGCs, are actually part of the ACD and to what extent they, and other ACD cells, contribute to the allantois and fetal tissues. To address these hypotheses, STELLA was immunolocalized to the mouse gastrula between Early Streak (ES) and 12-somite pair (-s) stages (~6.75-9.0 days post coitum, dpc) in histological sections. STELLA was found in both the nucleus and cytoplasm in a variety of cell types, both within and outside of the putative PGC trajectory. Fate-mapping the headfold-stage (~7.75-8.0 dpc) posterior region, by which time PGCs are thought to be segregated into a distinct lineage, revealed that the STELLA-positive proximal ACD and intraembryonic posterior primitive streak (IPS) contributed to a wide range of somatic tissues that encompassed derivatives of the three primary germ layers. This contribution included STELLA-positive cells localizing to tissues both within and outside of the putative PGC trajectory. Thus, while STELLA may identify a subpopulation of cells destined for the PGC lineage, our findings reveal that it may be part of a broader niche that encompasses the ACD and through which the STELLA population may contribute cells to a wide variety of posterior tissues of the mouse gastrula.

Lineage Commitments: Emphasis On Embryonic-Extraembryonic Interfaces.

The EMBO Workshop on 'Lineage Commitments: Emphasis on Embryonic-Extraembryonic Interfaces', held in May 2011, demonstrated that embryonic and extraembryonic tissues play early and significant interacting roles that mutually promote each other's further and correct deployment within the mammalian conceptus. Highlighted here are those presentations that directly addressed embryonic-extraembryonic interfaces in building the mammalian fetus.

Hedgehog signaling in the posterior region of the mouse gastrula suggests manifold roles in the fetal-umbilical connection and posterior morphogenesis.

Although many fetal birth defects, particularly those of the body wall and gut, are associated with abnormalities of the umbilical cord, the developmental relationship between these structures is largely obscure. Recently, genetic analysis of mid-gestation mouse embryos revealed that defects in Hedgehog signaling led to omphalocoele, or failure of the body wall to close at the umbilical ring (Matsumaru et al. [ 2011] PLos One 6:e16260). However, systematic spatiotemporal localization of Hedgehog signaling in the allantois, or umbilical precursor tissue, and the surrounding regions has not been documented. Here, a combination of reagents, including the Ptc1:lacZ and Runx1:lacZ reporter mice, immunohistochemistry for Smoothened (Smo), Sonic Hedgehog (Shh), and Indian hedgehog (Ihh), and detailed PECAM-1/Flk-1/Runx-1 analysis, revealed robust Hedgehog signaling in previously undocumented posterior sites over an extended period of time (∼7.0-9.75 dpc). These included the recently described proximal walls of the allantois (Ventral and Dorsal Cuboidal Mesothelia; VCM and DCM, respectively); the ventral embryonic surface continuous with them; hemogenic arterial endothelia; hematopoietic cells; the hindgut; ventral ectodermal ridge (VER); chorionic ectoderm; and the intraplacental yolk sac (IPY), which appeared to be a site of placental hematopoiesis. This map of Hedgehog signaling in the posterior region of the mouse conceptus will provide a valuable foundation upon which to elucidate the origin of many posterior midline abnormalities, especially those of the umbilical cord and associated fetal defects. Developmental Dynamics 240:2175-2193, 2011. © 2011 Wiley-Liss, Inc.

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

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'.

The enigmatic primitive streak: prevailing notions and challenges concerning the body axis of mammals.

The primitive streak establishes the antero-posterior body axis in all amniote species. It is thought to be the conduit through which mesoderm and endoderm progenitors ingress and migrate to their ultimate destinations. Despite its importance, the streak remains poorly defined and one of the most enigmatic structures of the animal kingdom. In particular, the posterior end of the primitive streak has not been satisfactorily identified in any species. Unexpectedly, and contrary to prevailing notions, recent evidence suggests that the murine posterior primitive streak extends beyond the embryo proper. In its extraembryonic site, the streak creates a node-like cell reservoir from which the allantois, a universal caudal appendage of all amniotes and the future umbilical cord of placental mammals, emerges. This new insight into the fetal/umbilical relationship may explain the etiology of a large number of umbilical-associated birth defects, many of which are correlated with abnormalities of the embryonic midline.

Collagen type IV and Perlecan exhibit dynamic localization in the Allantoic Core Domain, a putative stem cell niche in the murine allantois.

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.

Is extra-embryonic endoderm a source of placental blood cells?

The extra-embryonic hypoblast/visceral endoderm of Placentalia carries out a variety of functions during gestation, including hematopoietic induction. Results of decades-old and recent experiments have provided compelling evidence that, in addition to its inducing properties, hypoblast/visceral endoderm itself is a source of placental blood cells. Those observations that highlight extra-embryonic endoderm's role as an overlooked source of placental blood cells across species are briefly discussed here, with suggestions for future exploration.

Severing umbilical ties.

High levels of proteins called proteoglycans in the walls of umbilical arteries enable these arteries to close rapidly after birth and thus prevent blood loss in newborns.

The mouse fetal-placental arterial connection: A paradigm involving the primitive streak and visceral endoderm with implications for human development.

In Placentalia, the fetus depends upon an organized vascular connection with its mother for survival and development. Yet, this connection was, until recently, obscure. Here, we summarize how two unrelated tissues, the primitive streak, or body axis, and extraembryonic visceral endoderm collaborate to create and organize the fetal-placental arterial connection in the mouse gastrula. The primitive streak reaches into the extraembryonic space, where it marks the site of arterial union and creates a progenitor cell pool. Through contact with the streak, associated visceral endoderm undergoes an epithelial-to-mesenchymal transition, contributing extraembryonic mesoderm to the placental arterial vasculature, and to the allantois, or pre-umbilical tissue. In addition, visceral endoderm bifurcates into the allantois where, with the primitive streak, it organizes the nascent umbilical artery and promotes allantoic elongation to the chorion, the site of fetal-maternal exchange. Brachyury mediates streak extension and vascular patterning, while Hedgehog is involved in visceral endoderm's conversion to mesoderm. A unique CASPASE-3-positive cell separates streak- and non-streak-associated domains in visceral endoderm. Based on these new insights at the posterior embryonic-extraembryonic interface, we conclude by asking whether so-called primordial germ cells are truly antecedents to the germ line that segregate within the allantois, or whether they are placental progenitor cells. Incorporating these new working hypotheses into mutational analyses in which the placentae are affected will aid understanding a spectrum of disorders, including orphan diseases, which often include abnormalities of the umbilical cord, yolk sac, and hindgut, whose developmental relationship to each other has, until now, been poorly understood. This article is categorized under: Birth Defects > Associated with Preimplantation and Gastrulation Early Embryonic Development > Gastrulation and Neurulation.

Embryological origins of the human individual.

The human fertilized egg is not an embryo, but, more accurately, a conceptus, which contains all the information required to establish both the embryo and extraembryonic supporting tissues. The fertilized eggs of placental mammals, including humans, are entirely dependent upon the female's uterine environment for development to birth. At least half, possibly more, fertilized eggs, or potential lives, do not survive to birth, the greatest loss thought to occur during preimplantation and implantation. Which conceptuses will be lost and which will progress to birth cannot be predicted. The. preimplantation conceptus exhibits extreme developmental lability. Importantly, twinning can occur throughout the preimplantation and implantation phases, and thus, a single human individual has not emerged from the conceptus during this time period. Once the primitive streak is complete during early postimplantation development, identical twinning no longer occurs and the individual emerges. Thus, emergence of the human individual is a process. No single event thus far known is more important than any other prior to formation of the primitive streak. Formation of the streak is a defining moment in the origin of the individual. All further organization of the fetus occurs around this midline. Thus, by 14 days in the human, the body plan for life is established.

Generation of multipotent induced cardiac progenitor cells from mouse fibroblasts and potency testing in ex vivo mouse embryos.

Here we describe a protocol to generate expandable and multipotent induced cardiac progenitor cells (iCPCs) from mouse adult fibroblasts using forced expression of Mesp1, Tbx5, Gata4, Nkx2.5 and Baf60c (MTGNB) along with activation of Wnt and JAK/STAT signaling. This method does not use iPS cell factors and thus differs from cell activation and signaling-directed (CASD) reprogramming to cardiac progenitors. Our method is specific to direct CPC reprogramming, whereas CASD reprogramming can generate various cell types depending on culture conditions and raises the possibility of transitioning through a pluripotent cell state. The protocol describes how to isolate and infect primary fibroblasts; induce reprogramming and observe iCPC colonies; expand and characterize reprogrammed iCPCs by immunostaining, flow cytometry and gene expression; differentiate iCPCs in vitro into cardiac-lineage cells; and test the embryonic potency of iCPCs via injection into the cardiac crescent of mouse embryos. A scientist experienced in molecular cell biology and embryology can reproduce this protocol in 12-16 weeks. iCPCs can be used for studying cardiac biology, drug discovery and regenerative medicine.

Localization of Brachyury (T) in embryonic and extraembryonic tissues during mouse gastrulation.

T-box gene family members have important roles during murine embryogenesis, gastrulation, and organogenesis. Although relatively little is known about how T-box genes are regulated, published gene expression studies have revealed dynamic and specific patterns in both embryonic and extraembryonic tissues of the mouse conceptus. Mutant alleles of the T-box gene Brachyury (T) have identified roles in formation of mesoderm and its derivatives, such as somites and the allantois. However, given the cell autonomous nature of T gene activity and conflicting results of gene expression studies, it has been difficult to attribute a primary function to T in normal allantoic development. We report localization of T protein by sectional immunohistochemistry in both embryonic and extraembryonic tissues during mouse gastrulation, emphasizing T localization within the allantois. T was detected in all previously reported sites within the conceptus, including the primitive streak and its derivatives, nascent embryonic mesoderm, the node and notochord, as well as notochord-associated endoderm and posterior neurectoderm. In addition, we have clarified T within the allantois, where it was first detected in the proximal midline of the late allantoic bud (approximately 7.5 days postcoitum, dpc) and persisted within an expanded midline domain until 6-somite pairs (s; approximately 8.5 dpc). Lastly, we have discovered several novel T sites, including the developing heart, visceral endoderm, extraembryonic ectoderm, and its derivative, chorionic ectoderm. Together, these data provide a unified picture of T in the mammalian conceptus, and demonstrate T's presence in unrelated cell types and tissues in highly dynamic spatiotemporal patterns in both embryonic and extraembryonic tissues.