In the mouse, the first hematopoietic cells are generated in the yolk sac from the primitive, erythro-myeloid progenitor (EMP) and lymphoid programs that are specified before the emergence of hematopoietic stem cells. While many of the yolk sac-derived populations are transient, specific immune cell progeny seed developing tissues, where they function into adult life. To access the human equivalent of these lineages, we modeled yolk sac hematopoietic development using pluripotent stem cell differentiation. Here, we show that the combination of Activin A, BMP4, and FGF2 induces a population of KDR+CD235a/b+ mesoderm that gives rise to the spectrum of erythroid, myeloid, and T lymphoid lineages characteristic of the mouse yolk sac hematopoietic programs, including the Vδ2+ subset of γ/δ T cells that develops early in the human embryo. Through clonal analyses, we identified a multipotent hematopoietic progenitor with erythroid, myeloid, and T lymphoid potential, suggesting that the yolk sac EMP and lymphoid lineages may develop from a common progenitor.

The yolk sac (YS) is an extra-embryonic tissue that surrounds the yolk and absorbs, digests and transports nutrients during incubation of the avian embryo as well as during early term mammalian embryonic development. Understanding YS functions and development may enhance the efficient transfer of nutrients and optimize embryo development. To identify temporal large-scale patterns of gene expression and gain insights into processes and mechanisms in the YS, we performed a transcriptome study of the YS of chick embryos on embryonic days (E) E13, E15, E17, E19, and E21 (hatch).

The yolk sac tissue (YST) is a multifunctional metabolic organ supporting chicken embryonic development. This study examined whether incubation temperatures (ITs) affect YST functions. For this purpose, 300 eggs were assigned to 3 groups and incubated at control IT of 37.8°C, at 1.5°C below, 36.3°C (cold IT), and at 1.5°C above, 39.3°C (hot IT). For each group, 6 embryos' whole body mass and residual yolk (RSY) weights were recorded during incubation, and YST was sampled for both histology and gene expression analysis. YST functionality during incubation was examined by regression analysis, comparing changes in expression patterns of genes involved in lipid uptake and metabolism (LRP2, ApoA1), oligopeptides uptake (PepT1), gluconeogenesis (FBP1), glycogenesis (GYS2), and thyroid hormones regulation (TTR, DIO1, DIO2). Results show that hot and cold ITs affected YST gene expression and yolk utilization. PepT1 expression decreased towards hatch, in both hot and cold ITs, while in the Control IT, it reached a plateau. ApoA1 and DIO2 expression showed a moderate linear fit compared to polynomial fit in the control. GYS2 expression had no change along incubation, while in the control IT, it showed a polynomial fit. Expression of LRP2, FBP1, and DIO1 genes was affected by either cold or hot IT's. TTR expression patterns were similar in all IT groups. The variations in gene expression patterns observed in the 3 ITs can explain the changes in yolk utilization, an important parameter for hatchling quality. While the control IT showed optimal utilization, with an RSY value of 11.12% at the day of hatch, the cold and hot IT groups exhibited lower utilization with an RSY value of 18.18 and 29.99%, respectively. These findings are the first to show that ITs change the expression of key YST genes, leading to variations in yolk utilization by the embryo.

Implantation of the human embryo begins a critical developmental stage that comprises profound events including axis formation, gastrulation and the emergence of haematopoietic system1,2. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons3-5. Stem cell models of human embryo have emerged to help unlock the mysteries of this stage6-16. Here we present a genetically inducible stem cell-derived embryoid model of early post-implantation human embryogenesis that captures the reciprocal codevelopment of embryonic tissue and the extra-embryonic endoderm and mesoderm niche with early haematopoiesis. This model is produced from induced pluripotent stem cells and shows unanticipated self-organizing cellular programmes similar to those that occur in embryogenesis, including the formation of amniotic cavity and bilaminar disc morphologies as well as the generation of an anterior hypoblast pole and posterior domain. The extra-embryonic layer in these embryoids lacks trophoblast and shows advanced multilineage yolk sac tissue-like morphogenesis that harbours a process similar to distinct waves of haematopoiesis, including the emergence of erythroid-, megakaryocyte-, myeloid- and lymphoid-like cells. This model presents an easy-to-use, high-throughput, reproducible and scalable platform to probe multifaceted aspects of human development and blood formation at the early post-implantation stage. It will provide a tractable human-based model for drug testing and disease modelling.

Yolk-sac tumours (YSTs), a germ cell tumour subtype, occur in newborns and infants as well as in young adults of age 14-44 years. In clinics, adult patients with YSTs face a poor prognosis, as these tumours are often therapy-resistant and count for many germ cell tumour related deaths. So far, the molecular and (epi)genetic mechanisms that control development of YST are far from being understood. We deciphered the molecular and (epi)genetic mechanisms regulating YST formation by meta-analysing high-throughput data of gene and microRNA expression, DNA methylation and mutational burden. We validated our findings by qRT-PCR and immunohistochemical analyses of paediatric and adult YSTs. On a molecular level, paediatric and adult YSTs were nearly indistinguishable, but were considerably different from embryonal carcinomas, the stem cell precursor of YSTs. We identified FOXA2 as a putative key driver of YST formation, subsequently inducing AFP, GPC3, APOA1/APOB, ALB and GATA3/4/6 expression. In YSTs, WNT-, BMP- and MAPK signalling-related genes were up-regulated, while pluripotency- and (primordial) germ cell-associated genes were down-regulated. Expression of FOXA2 and related key factors seems to be regulated by DNA methylation, histone methylation / acetylation and microRNAs. Additionally, our results highlight FOXA2 as a promising new biomarker for paediatric and adult YSTs.

The yolk sac is the first site of blood-cell production during embryonic development in both murine and human. Heat shock proteins (HSPs), including HSP70 and HSP27, have been shown to play regulatory roles during erythropoiesis. However, it remains unknown whether HSP60, a molecular chaperone that resides mainly in mitochondria, could also regulate early erythropoiesis. In this study, we used Tie2-Cre to deactivate the Hspd1 gene in both hematopoietic and vascular endothelial cells, and found that Tie2-Cre+Hspd1f/f (HSP60CKO) mice were embryonic lethal between the embryonic day 10.5 (E10.5) and E11.5, exhibiting growth retardation, anemia, and vascular defects. Of these, anemia was observed first, independently of vascular and growth phenotypes. Reduced numbers of erythrocytes, as well as an increase in cell apoptosis, were found in the HSP60CKO yolk sac as early as E9.0, indicating that deletion of HSP60 led to abnormality in yolk sac erythropoiesis. Deletion of HSP60 was also able to reduce mitochondrial membrane potential and the expression of the voltage-dependent anion channel (VDAC) in yolk sac erythrocytes. Furthermore, cyclosporine A (CsA), which is a well-recognized modulator in regulating the opening of the mitochondrial permeability transition pore (mPTP) by interacting with Cyclophilin D (CypD), could significantly decrease cell apoptosis and partially restore VDAC expression in mutant yolk sac erythrocytes. Taken together, we demonstrated an essential role of HSP60 in regulating yolk sac cell survival partially via a mPTP-dependent mechanism.

CRISPR-mediated genome editing in vivo can be accompanied by prolonged stability of the Cas9 protein in mouse embryos. Then, genome edited variant alleles will be induced as long as Cas9 protein is active, and unmodified wildtype target loci are available. The corollary is that CRISPR-modified alleles that arise after the first zygotic cell division potentially could be distributed asymmetrically to the cell lineages that are specified early during morula and blastocyst development. This has practical implications for the investigation of F0 generation individuals, as cells in embryonic and extraembryonic tissues, such as the visceral yolk sac, might end up inheriting different genotypes. We here investigated the hypothetically possible scenarios by genotyping individual F0 CRISPants and their associated visceral yolk sacs in parallel. In all cases, we found that embryonic genotype was accurately reflected by yolk sac genotyping, with the two tissues indicating genetic congruence, even when the conceptus was a mosaic of cells with distinct allele configurations. Nevertheless, low abundance of a variant allele may represent a private mutation occurring only in the yolk sac, and in those rare cases, additional genotyping to determine the mutational status of the embryo proper is warranted.

Introduction: Maternal diabetes during pregnancy is well known to be associated with a higher risk for structural birth defects in the offspring. Recent searches for underlying mechanisms have largely focused on aberrant processes in the embryo itself, although prior research in rodent models implicated dysfunction also of the visceral yolk sac. The objective of our research was to investigate both tissues within the conceptus simultaneously. Methods: We conducted unbiased transcriptome profiling by RNA sequencing on pairs of individual yolk sacs and their cognate embryos, using the non-obese diabetic (NOD) mouse model. The analysis was performed at gestational day 8.5 on morphologically normal specimen to circumvent confounding by defective development. Results: Even with large sample numbers (n = 33 in each group), we observed considerable variability of gene expression, primarily driven by exposure to maternal diabetes, and secondarily by developmental stage of the embryo. Only a moderate number of genes changed expression in the yolk sac, while in the embryo, the exposure distinctly influenced the relationship of gene expression levels to developmental progression, revealing a possible role for altered cell cycle regulation in the response. Also affected in embryos under diabetic conditions were genes involved in cholesterol biosynthesis and NAD metabolism pathways. Discussion: Exposure to maternal diabetes during gastrulation changes transcriptomic profiles in embryos to a substantially greater effect than in the corresponding yolk sacs, indicating that despite yolk sac being of embryonic origin, different mechanisms control transcriptional activity in these tissues. The effects of maternal diabetes on expression of many genes that are correlated with developmental progression (i.e. somite stage) highlight the importance of considering developmental maturity in the interpretation of transcriptomic data. Our analyses identified cholesterol biosynthesis and NAD metabolism as novel pathways not previously implicated in diabetic pregnancies. Both NAD and cholesterol availability affect a wide variety of cellular signaling processes, and can be modulated by diet, implying that prevention of adverse outcomes from diabetic pregnancies may require broad interventions, particularly in the early stages of pregnancy.

The chicken yolk sac (YS) plays an important role in nutrient absorption and immune function for the developing embryo. The avian β-defensins (AvBD) are cationic peptides that are important members of the innate immune system. The objective of this study was to profile AvBD mRNA expression patterns and distribution of cells expressing AvBD mRNA in the chicken YS. Expression of AvBD1, 2, 7, and 10 mRNA was low at embryonic day 7 (e7), increased to e9 through e13 and then declined to e19. Using in situ hybridization, AvBD10 mRNA was found to be expressed in endodermal epithelial cells, while AvBD1, 2, and 7 mRNA were expressed in heterophils. The developmental expression pattern and distribution of AvBD mRNA in the YS reveals the importance of these genes to protection of the developing chick embryo.

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

Regenerative medicine has been growing because of the emergent need for tissues/organs for transplants and restorative surgeries. Biological scaffolds are important tools to try to solve this problem. The one used in this reserach was developed by an acellular biological scaffold from canine placenta with a rich source of cellular matrix. After decellularization, the cellular matrix demonstrated structural preservation with the presence of important functional proteins such as collagen, fibronectin, and laminin. We used cells transduced with vascular endothelial growth factor (VEGF) to recellularize this scaffold. It was succeeded by seeding the cells in nonadherent plaques in the presence of the sterelized placenta scaffold. Cells were adhered to the scaffold when analyzed by immunocytochemistry and scanning electron microscopy, both showing sprouting of yolk sac VEGF (YSVEGF) cells. This recellularized scaffold is a promissory biomaterial for repairing injured areas where neovascularization is required.

Renal macrophages represent a highly heterogeneous and specialized population of myeloid cells with mixed developmental origins from the yolk-sac and hematopoietic stem cells (HSC). They promote both injury and repair by regulating inflammation, angiogenesis, and tissue remodeling. Recent reports highlight differential roles for ontogenically distinct renal macrophage populations in disease. However, little is known about how these populations change over time in normal, uninjured kidneys. Prior reports demonstrated a high proportion of HSC-derived macrophages in the young adult kidney. Unexpectedly, using genetic fate-mapping and parabiosis studies, we found that yolk-sac-derived macrophages progressively expand in number with age and become a major contributor to the renal macrophage population in older mice. This chronological shift in macrophage composition involves local cellular proliferation and recruitment from circulating progenitors and may contribute to the distinct immune responses, limited reparative capacity, and increased disease susceptibility of kidneys in the elderly population.

The signaling cascades that direct the morphological differentiation of the vascular system during early embryogenesis are not well defined. Several signaling pathways, including Notch and VEGF signaling, are critical for the formation of the vasculature in the mouse. To further understand the role of Notch signaling during endothelial differentiation and the genes regulated by this pathway, both loss-of-function and gain-of-function approaches were analyzed in vivo.

It is known that the adhesion molecule ALCAM (CD166) mediates metastasis of malignant cells and organogenesis in embryos. We show here that embryonic day 8.5 (E8.5) murine yolk sac cells express ALCAM protein and that ALCAM expression can be used to define endothelial and cardiac precursors from hematopoietic precursors in E8.5 yolk sacs. ALCAM high+ cells exclusively give rise to endothelial and cardiac cells in matrigel assays but generate no hematopoietic colonies in methylcellulose assays. ALCAM low+ and ALCAM- populations predominantly give rise to hematopoietic cells in methylcellulose, but do not generate any cell clusters in matrigel. The ALCAM high+ population contains both Flk-1+ and Flk-1- cells. The former population exclusively contains endothelial cells whereas the latter give rise to cardiac cells when cultured on OP9 stromal cells. We also show that cardiac lineage marker genes such as Nkx-2.5, and the endothelial marker VE-cadherin are expressed in the ALCAM high+ fraction, whereas the hematopoietic marker GATA1 and Runx1 are expressed in the ALCAM low+/- fraction. However, we did not detect expression of the cardiac structural protein cTn-T in cells from yolk sac cells until these had had been differentiated on OP9 for 5 days. Altogether, these results indicate that cells retaining a potential to differentiate to the cardiac lineage are present in E8.5 yolk sacs and can be isolated as ALCAM high+, Flk-1- cells. Our report provides novel insights into the origin and differentiation process of cardiac cells in the formation of the circulatory system.

Human embryogenesis entails complex signalling interactions between embryonic and extra-embryonic cells. However, how extra-embryonic cells direct morphogenesis within the human embryo remains largely unknown due to a lack of relevant stem cell models. Here, we have established conditions to differentiate human pluripotent stem cells (hPSCs) into yolk sac-like cells (YSLCs) that resemble the post-implantation human hypoblast molecularly and functionally. YSLCs induce the expression of pluripotency and anterior ectoderm markers in human embryonic stem cells (hESCs) at the expense of mesoderm and endoderm markers. This activity is mediated by the release of BMP and WNT signalling pathway inhibitors, and, therefore, resembles the functioning of the anterior visceral endoderm signalling centre of the mouse embryo, which establishes the anterior-posterior axis. Our results implicate the yolk sac in epiblast cell fate specification in the human embryo and propose YSLCs as a tool for studying post-implantation human embryo development in vitro.

Several identified genes play key roles in the specification of the blood-forming system, from commitment of mesoderm to differentiation of hemopoietic and endothelial cells. We have thoroughly analyzed the expression dynamics of some of these genes during yolk sac erythropoiesis in the chick embryo. The study includes transcription factors which are known to participate in multimeric complexes: GATA-1, -2, SCL/tal-1 and Lmo2 (whose avian orthologue we have cloned), VEGF-R2, a critical regulator of hemopoietic and endothelial commitment, and hemoglobin used as a marker of the last step in erythroid differentiation. Several findings were unexpected. (1) Two distinct patterns were revealed for GATA-2, first: low expression, ubiquitous in all mesodermal cells, as soon as cells ingress through the primitive streak; secondly: high, blood island-specific expression. (2) VEGF-R2 is coexpressed with GATA-2 at the level of the primitive streak. (3) SCL and Lmo2 expression is restricted to presumptive hemangioblasts. (4) The up-regulation of GATA-2 in newly formed blood islands is shortly followed by GATA-1 expression. (5) Lmo2 is up-regulated in blood island angioblasts thus appearing as one of the earliest markers for endothelial cell commitment. VEGF-R2 is down-regulated in hemopoietic cells prior to GATA-2, SCL/tal-1, Lmo2 and GATA-1 in erythroblasts.

Tissue macrophages in many adult organs originate from yolk sac (YS) progenitors, which invade the developing embryo and persist by means of local self-renewal. However, the route and characteristics of YS macrophage trafficking during embryogenesis are incompletely understood. Here we show the early migration dynamics of YS-derived macrophage progenitors in vivo using fate mapping and intravital microscopy. From embryonic day 8.5 (E8.5) CX3CR1+ pre-macrophages are present in the mouse YS where they rapidly proliferate and gain access to the bloodstream to migrate towards the embryo. Trafficking of pre-macrophages and their progenitors from the YS to tissues peaks around E10.5, dramatically decreases towards E12.5 and is no longer evident from E14.5 onwards. Thus, YS progenitors use the vascular system during a restricted time window of embryogenesis to invade the growing fetus. These findings close an important gap in our understanding of the development of the innate immune system.

Accurately determining the normal range of early pregnancy markers can help to predict adverse pregnancy outcomes. The variance in ovulation days leads to uncertain accuracy of reference intervals for natural pregnancies. While the gestational age (GA) is accurate estimation during in vitro fertilization-embryo transfer (IVF-ET). Thus, the objective of this research is to construct reference intervals for gestational sac diameter (GSD), yolk sac diameter (YSD), embryonic length (or crown-rump length, CRL) and embryonic heart rate (HR) at 6-10 gestational weeks (GW) after IVF-ET.

Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM. Eomes regulates the accessibility of enhancers that the transcription factor stem cell leukaemia (SCL) normally utilizes to specify primitive erythrocytes and is essential for the normal development of Runx1+ haemogenic endothelium. Single-cell RNA sequencing suggests that Eomes loss of function profoundly blocks the formation of blood progenitors but not specification of Flk-1+ haematoendothelial progenitors. Our findings place Eomes at the top of the transcriptional hierarchy regulating early blood formation and suggest that haemogenic competence is endowed earlier during embryonic development than was previously appreciated.

Tissue-resident macrophages have mixed developmental origins. They derive in variable extent from yolk sac (YS) hematopoiesis during embryonic development. Bone marrow (BM) hematopoietic progenitors give rise to tissue macrophages in postnatal life, and their contribution increases upon organ injury. Since the phenotype and functions of macrophages are modulated by the tissue of residence, the impact of their origin and developmental paths has remained incompletely understood.