Karen M. Downs

The allantois and chorion, when isolated before circulation or chorio-allantoic fusion, have hematopoietic potential

The chorio-allantoic placenta forms through the fusion of the allantois (progenitor tissue of the umbilical cord), with the chorionic plate. The murine placenta contains high levels of hematopoietic stem cells, and is therefore a stem cell niche. However, it is not known whether the placenta is a site of hematopoietic cell emergence, or whether hematopoietic cells originate from other sites in the conceptus and then colonize the placenta. Here, we show that the allantois and chorion, isolated prior to the establishment of circulation, have the potential to give rise to myeloid and definitive erythroid cells following explant culture. We further show that the hematopoietic potential of the allantois and chorion does not require their union, indicating that it is an intrinsic property of these tissues. These results suggest that the placenta is not only a niche for, but also a source of, hematopoietic cells.

Contrasting patterns of myc and N-myc expression during gastrulation of the mouse embryo.

myc and N-myc are related genes whose similar protein products may be used for different purposes in vertebrate organisms. We have explored this possibility by using hybridization in situ to examine the expression of myc and N-myc during gastrulation of mouse embryos. Throughout gastrulation, myc RNA was most abundant in extraembryonic cells; by contrast, N-myc RNA was found at highest levels in the expanding primitive streak and other portions of the embryonic mesoderm. Differentiation of mesoderm to epithelioid cells was accompanied by diminished expression of N-myc. Expression of myc was not an inevitable correlate of cellular proliferation. Instead, the gene appeared to be regulated in concert with changes that affect a diversity of cellular properties, including proliferation, invasiveness, and differentiation.

Functional ablation of the mouse Ldb1 gene results in severe patterning defects during gastrulation

The LIM domain-binding protein 1 (Ldb1) is found in multi-protein complexes containing various combinations of LIM-homeodomain, LIM-only, bHLH, GATA and Otx transcription factors. These proteins exert key functions during embryogenesis. Here we show that targeted deletion of the Ldb1 gene in mice results in a pleiotropic phenotype. There is no heart anlage and head structures are truncated anterior to the hindbrain. In about 40% of the mutants, posterior axis duplication is observed. There are also severe defects in mesoderm-derived extraembryonic structures, including the allantois, blood islands of the yolk sack, primordial germ cells and the amnion. Abnormal organizer gene expression during gastrulation may account for the observed axis defects in Ldb1 mutant embryos. The expression of several Wnt inhibitors is curtailed in the mutant, suggesting that Wnt pathways may be involved in axial patterning regulated by Ldb1.

Erythroid Krüppel‐like factor exhibits an early and sequentially localized pattern of expression during mammalian erythroid ontogeny

Erythroid Krüppel‐like factor (EKLF) is an erythroid cell‐specific transcription factor that mediates activation via binding to a 9 base pair sequence that encompasses the CACCC element, one of a trio of evolutionarily conserved sequence motifs that are functionally important for transcription of red cell‐specific genes. Molecular analyses have delineated the specificity of its interaction and activation through the CAC site at the adult β‐globin promoter. However, its expression and distribution during murine ontogeny have not been established. To address these issues, we have focused on biological aspects of EKLF expression by examining the onset and localization of its mRNA during murine development by using reverse transcription/polymerase chain reaction (RT/PCR) analysis of differentiating embryonic stem cells and in situ analyses of normal developing embryos. In addition, we have monitored the presence of EKLF protein by blot analysis of whole‐cell extracts derived from circulating cells and embryonic tissue.

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.

Lineage Reprogramming of Fibroblasts into Proliferative Induced Cardiac Progenitor Cells by Defined Factors

Several studies have reported reprogramming of fibroblasts into induced cardiomyocytes; however, reprogramming into proliferative induced cardiac progenitor cells (iCPCs) remains to be accomplished. Here we report that a combination of 11 or 5 cardiac factors along with canonical Wnt and JAK/STAT signaling reprogrammed adult mouse cardiac, lung, and tail tip fibroblasts into iCPCs. The iCPCs were cardiac mesoderm-restricted progenitors that could be expanded extensively while maintaining multipotency to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells in vitro. Moreover, iCPCs injected into the cardiac crescent of mouse embryos differentiated into cardiomyocytes. iCPCs transplanted into the post-myocardial infarction mouse heart improved survival and differentiated into cardiomyocytes, smooth muscle cells, and endothelial cells. Lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for drug discovery, disease modeling, and cardiac regenerative therapy.

Systematic localization of oct‐3/4 to the gastrulating mouse conceptus suggests manifold roles in mammalian development

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‐3/4s expression profile suggests unexpected and potentially far more versatile roles in development than have been previously appreciated. Developmental Dynamics 237:464–475, 2008.

Brachyury is required for elongation and vasculogenesis in the murine allantois.

Mouse conceptuses homozygous for mutations in brachyury (T) exhibit a short, misshapen allantois that fails to fuse with the chorion. Ultimately, mutant embryos die during mid-gestation. In the 60 years since this discovery, the role of T in allantoic development has remained obscure. T protein was recently identified in several new sites during mouse gastrulation, including the core of the allantois, where its function is not known. Here, using molecular, genetic and classical techniques of embryology, we have investigated the role of T in allantoic development. Conceptuses homozygous for the TCurtailed (TC) mutation (TC/TC) exhibited allantoic dysmorphogenesis shortly after the allantoic bud formed. Diminution in allantoic cell number and proliferation was followed by cell death within the core. Fetal liver kinase (Flk1)-positive angioblasts were significantly decreased in TC/TC allantoises and did not coalesce into endothelial tubules, possibly as a result of the absence of platelet endothelial cell adhesion molecule 1 (Pecam1), whose spatiotemporal relationship to Flk1 suggested a role in patterning the umbilical vasculature. Remarkably, microsurgical perturbation of the wild-type allantoic core phenocopied the TC/TC vascularization defect, providing further support that an intact core is essential for vascularization. Last, abnormalities were observed in the TC/TC heart and yolk sac, recently reported sites of T localization. Our findings reveal that T is required to maintain the allantoic core, which is essential for allantoic elongation and vascular patterning. In addition, morphological defects in other extraembryonic and embryonic vascular organs suggest a global role for T in vascularization of the conceptus.

Investigation into a role for the primitive streak in development of the murine allantois.

Despite its importance as the source of one of three major vascular systems in the mammalian conceptus, little is known about the murine allantois, which will become the umbilical cord of the chorio-allantoic placenta. During gastrulation, the allantois grows into the exocoelomic cavity as a mesodermal extension of the posterior primitive streak. On the basis of morphology, gene expression and/or function, three cell types have been identified in the allantois: an outer layer of mesothelial cells, whose distal portion will become transformed into chorio-adhesive cells, and endothelial cells within the core. Formation of endothelium and chorio-adhesive cells begins in the distal region of the allantois, farthest from the streak. Over time, endothelium spreads to the proximal allantoic region, whilst the distal outer layer of presumptive mesothelium gradually acquires vascular cell adhesion molecule (VCAM1) and mediates chorio-allantoic union. Intriguingly, the VCAM1 domain does not extend into the proximal allantoic region. How these three allantoic cell types are established is not known, although contact with the chorion has been discounted. In this study, we have investigated how the allantois differentiates, with the goal of discriminating between extrinsic mechanisms involving the primitive streak and an intrinsic role for the allantois itself. Exploiting previous observations that the streak contributes mesoderm to the allantois throughout the latters early development, microsurgery was used to remove allantoises at ten developmental stages. Subsequent whole embryo culture of operated conceptuses resulted in the formation of regenerated allantoises at all time points. Aside from being generally shorter than normal, none of the regenerates exhibited abnormal differentiation or inappropriate cell relationships. Rather, all of them resembled intact allantoises by morphological, molecular and functional criteria. Moreover, fate mapping adjacent yolk sac and amniotic mesoderm revealed that these tissues and their associated bone morphogenetic protein 4 (BMP4) did not contribute to restoration of allantoic outgrowth and differentiation during allantoic regeneration. Thus, on the basis of these observations, we conclude that specification of allantoic endothelium, mesothelium and chorio-adhesive cells does not occur by a streak-related mechanism during the time that proximal epiblast travels through it and is transformed into allantoic mesoderm. Rather, all three cell-types are established by mechanisms intrinsic to the allantois, and possibly include roles for cell age and cell position. However, although chorio-adhesive cells were not specified within the streak, we discovered that the streak nonetheless plays a role in establishing VCAM1s expression domain, which typically began and was thereafter maintained at a defined distance from the primitive streak. When allantoises were removed from contact with the streak, normally VCAM1-negative proximal allantoic regions acquired VCAM1. These results suggested that the streak suppresses formation of chorio-adhesive cells in allantoic mesoderm closest to it. Together with previous results, findings presented here suggest a model of differentiation of allantoic mesoderm that invokes intrinsic and extrinsic mechanisms, all of which appear to be activated once the allantoic bud has formed.

1 The Murine Allantois

Publisher Summary This chapter focuses on the development of the mouse allantois. The murine allantois is the future umbilical component of the chorioallantoic placenta. Its primary function is to fuse with the chorion and vascularize, thereby serving as the vital connection between mother and fetus for the interchange of nutrients, gases, and metabolic wastes during much of mammalian gestation. Whereas, much effort has been devoted to investigating the role of the mature placenta, studies of the development of its component structures, the chorion and the allantois, have been minimal. In the development of any vertebrate embryo, only part of the eggs cleavage cell mass will form the actual embryo; the other part will elaborate the extraembryonic structures, referred to—especially in mammals—as “fetal membranes.” The set of fetal membranes comprises the yolk sac, amnion, chorion, and allantois. Fetal membranes are shed at birth by natural methods and discarded. The allantois may provide fresh approaches to old problems. Because the principal function of the allantois is to vascularize, the allantois is a very attractive organ for the study of vasculogenesis, especially as it can be isolated free of contamination from embryonic and extraembryonic tissues.