Multiple cell populations generate macrophage progenitors in the early yolk sac.

Yolk sac (YS) CSF1 receptor positive (CSF1R+) cells are thought to be the progenitors for tissue-resident macrophages present in various tissues. The YS progenitors for tissue-resident macrophages are referred to as erythroid-myeloid progenitors (EMPs). However, diverse types of hematopoietic progenitors are present in the early YS, thus it is not precisely known which type of hematopoietic cell gives rise to the CSF1R+ lineage. In this study, an analysis was conducted to determine when CSF1R+ progenitors appeared in the early YS. It showed that CSF1R+ cells appeared in the YS as early as embryonic day 9 (E9) and that the earliest hematopoietic progenitors that differentiate into CSF1R+ cells were found in E8. Since these progenitors possessed the capability to generate primitive erythroid cells, it was likely that primitive erythroid lineages shared progenitors with the CSF1R+ lineage. Mutual antagonism appears to work between PU.1 and GATA1 when CSF1R+ cells appear in the early YS. One day later (E9), multiple progenitors, including myeloid-restricted progenitors and multipotent progenitors, in the YS could immediately generate CSF1R+ cells. These results suggest that EMPs are not an exclusive source for the CSF1R+ lineage; rather, multiple hematopoietic cell populations give rise to CSF1R+ lineage in the early YS.

Interaction of extraembryonic microglia and neonatal brain development.

Microglia are resident immune cells in the central nervous system (CNS), which, in a healthy state, promote CNS homeostasis and respond to CNS injury. In contrast, microglia are also implicated in pathological conditions where they may contribute to neural damage. Primitive microglia arise from extraembryonic progenitors in the yolk sac (YS). The extraembryonic origins of primitive microglia are distinct from other tissue macrophages. The YS is the first site of hematopoiesis in development. Uniquely, microglial pregenital cells in the mouse derive from an early myeloid branch of the hematopoietic lineage in the YS. Microglia are critical in several key stages of physiological brain development, including embryonic vasculogenesis, immunosurveillance, and neurogenesis. Abnormal microglial function has been linked to neurodevelopmental and neurodegenerative diseases, although mechanistic roles in disease etiology remain incompletely understood. Knowledge of species-specific differences between human, murine and other animal models is also critical to understanding translational relevance to human health and disease as biomedical understanding of the importance of primitive microglia advances. This significance drives the importance of understanding, comparatively, the extraembryonic origins and developmental mechanisms whereby human primitive microglia differentiate and migrate to inform translational research. A better understanding of the molecular drivers may lead to biomarkers and/or preventative or therapeutic measures for neonatal brain development and neurodegenerative diseases. Herein, the role of microglia in neonatal brain development is discussed, current understandings of the developmental origins of microglia are described, the ontogeny and phylogeny of microglia, and implications of in vitro microglia-like cell differentiation, with a specific interest on neurodegenerative diseases, are reviewed. Together, these emphasize the importance of leveraging the extraembryonic origins of microglia to not only better understand neurodevelopment and neurodegenerative diseases, but also to develop protective measures that are specific to human microglia.

Centennial Review: The chicken yolk sac is a multifunctional organ.

The yolk sac (YS) consists of the yolk, which supplies nutrients, and the YS tissue, which surrounds the yolk and provides essential metabolic functions for the developing embryo. The YS tissue is derived from the midgut of the embryo and consists of a layer of endodermal epithelial cells (EEC) in contact with the yolk contents, a mesodermal layer that contains the vascular system and an outer ectodermal layer. The YS tissue is a multifunctional organ that provides essential functions such as host immunity, nutrient uptake, carbohydrate and lipid metabolism, and erythropoiesis. The YS tissue plays a role in immunity by the transport of maternal antibodies in the yolk to the embryonic circulation that feeds the developing embryo. In addition, the YS tissue expresses high mRNA levels of the host defense peptide, avian ß-defensin 10 during mid embryogenesis. Owing to its origin, the YS EEC share some functional properties with intestinal epithelial cells such as expression of transporters for amino acids, peptides, monosaccharides, fatty acids, and minerals. The YS tissue stores glycogen and expresses enzymes for glycogen synthesis and breakdown and glucogenesis. This carbohydrate metabolism may play an important role in the hatching process. The mesodermal layer of the YS tissue is the site for erythropoiesis and provides erythrocytes before the maturation of the bone marrow. Other functions of the YS tissue involve synthesis of plasma proteins, lipid transport and cholesterol metabolism, and synthesis of thyroxine. Thus, the YS is an essential organ for the growth, development, and health of the developing embryo. This review will provide an overview of the studies that have investigated the functionalities of the YS tissue at the cellular and molecular levels with a focus on chickens.

Yolk sac, but not hematopoietic stem cell-derived progenitors, sustain erythropoiesis throughout murine embryonic life.

In the embryo, the first hematopoietic cells derive from the yolk sac and are thought to be rapidly replaced by the progeny of hematopoietic stem cells. We used three lineage-tracing mouse models to show that, contrary to what was previously assumed, hematopoietic stem cells do not contribute significantly to erythrocyte production up until birth. Lineage tracing of yolk sac erythromyeloid progenitors, which generate tissue resident macrophages, identified highly proliferative erythroid progenitors that rapidly differentiate after intra-embryonic injection, persisting as the major contributors to the embryonic erythroid compartment. We show that erythrocyte progenitors of yolk sac origin require 10-fold lower concentrations of erythropoietin than their hematopoietic stem cell-derived counterparts for efficient erythrocyte production. We propose that, in a low erythropoietin environment in the fetal liver, yolk sac-derived erythrocyte progenitors efficiently outcompete hematopoietic stem cell progeny, which fails to generate megakaryocyte and erythrocyte progenitors.

Yolk sac hematopoiesis: does it contribute to the adult hematopoietic system?

In most vertebrates, the yolk sac (YS) represents the very first tissue where blood cells are detected. Therefore, it was thought for a long time that it generated all the blood cells present in the embryo. This model was challenged using different animal models, and we now know that YS hematopoietic precursors are mostly transient although their contribution to the adult system cannot be excluded. In this review, we aim at properly define the different waves of blood progenitors that are produced by the YS and address the fate of each of them. Indeed, in the last decade, many evidences have emphasized the role of the YS in the emergence of several myeloid tissue-resident adult subsets. We will focus on the development of microglia, the resident macrophages in the central nervous system, and try to untangle the recent controversy about their origin.

In ovo injection of branched-chain amino acids: Embryonic development, hatchability and hatching quality of turkey poults.

In this study, the influence of a branched-chain amino acid blend (BCAA composed of 3 l-leucine:1 l-valine:2 l-isoleucine) injected into the amniotic fluid was evaluated for embryonic growth, yolk-sac (YS) utilization and development of gastrointestinal tract (GIT) and skeletal muscles of turkey embryos from day 24 of incubation (24E) to hatching, together with hatchability, poult quality and liver L* (lightness), a* (redness) and b* (yellowness) values at hatch. At day 22 of incubation, embryonated eggs (n = 240) were assigned to three treatments, that is, eggs were not injected (control, NC) or injected with 1.5 ml sterile solution with 0.9% salt (SA) or 0.2% BCAA blend (BCAAb). These solutions were injected manually into the amniotic fluid of the embryonated eggs. To determine weights and lengths (where appropriate) of the studied organs and tissues, four embryonated eggs and poults per treatment were selected at 24E and at hatch. While the BCAAb decreased the YS and embryo weight, hatchability and the liver L* value, it increased the weight and quality of poults and the weights of breast and thigh muscles at hatch. In conclusion, the in ovo feeding of the BCAA blend negatively affected hatchability but positively affected hatching weight and poult quality by improving development of skeletal muscles and by regulating energy metabolism.

Omega-3 polyunsaturated fatty acids promote angiogenesis in placenta derived mesenchymal stromal cells.

Enhancement of angiogenesis is solicited in wound repair and regeneration. Mesenchymal stromal cells derived from the placenta (P-MSCs) have an inherent angiogenic potential. Polyunsaturated fatty acids (PUFAs) in turn, specifically the omega-3 (N-3) are essential for growth and development. They are also recommended as dietary supplements during pregnancy. We therefore hypothesized that addition of N-3 PUFAs in P-MSC culture media may enhance their angiogenic potential. Hence, we treated P-MSCs with omega-3 (N-3) fatty acids -Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) at different concentrations and tested their angiogenic potential. We saw an upregulation of both bFGF and VEGFA. We also found enhanced in vitro tube formation ability of P-MSCs treated with DHA: EPA. We then looked at the influence of the conditioned medium (CM) collected from P-MSCs exposed to DHA: EPA on the key effector cells -HUVECs (Human Umbilical Vein derived endothelial cells and their functionality was further confirmed on chick yolk sac membrane. We found that the CM of P-MSCs exposed to DHA: EPA could enhance angiogenesis in both cases. These result were finally validated in an in vivo matrigel plug assay which revealed enhanced migration and vessel formation in CM treated with DHA: EPA. Our data thus reveals for the first time that supplementation with lower concentration of PUFA enhances the angiogenic potential of P-MSCs making them suitable for chronic wound healing applications.

Transdifferentiation of mouse visceral yolk sac cells into parietal yolk sac cells in vitro.

The mouse embryonic yolk sac is an extraembryonic membrane that consists of a visceral yolk sac (VYS) and parietal yolk sac (PYS), and functions in hematopoietic-circulation in the fetal stage. The present study was undertaken to examine the normal development of both murine VYS and PYS tissues using various molecular markers, and to establish a novel VYS cell culture system in vitro for analyzing differentiation potentials of VYS cells. RT-PCR and immunohistochemical analyses of gene expression in VYS and PYS tissues during development revealed several useful markers for their identification: HNF1ß, HNF4α, Cdh1 (E-cadherin), Krt8 and Krt18 for VYS epithelial cells, and Stra6, Snail1, Thbd and vimentin for PYS cells. PYS cells exhibited mesenchymal characteristics in gene expression and morphology. When VYS cells at 11.5 days of gestation were cultured in vitro for 7 days, the number of HNF1ß-, HNF4α-, E-cadherin- and cytokeratin-positive VYS epithelial cells was significantly reduced and, instead, Stra6-and vimentin-positive PYS-like cells increased with culture. RT-PCR analyses also demonstrated that gene expression of VYS markers decreased, whereas that of PYS markers increased in the primary culture of VYS cells. These data indicate that VYS epithelial cells rapidly transdifferentiate into PYS cells having mesenchymal characteristics in vitro, which may provide a culture system suitable for studying molecular mechanisms of VYS transdifferentiation into PYS cells and also epithelial-mesenchymal transition.

Most Tissue-Resident Macrophages Except Microglia Are Derived from Fetal Hematopoietic Stem Cells.

Macrophages are one of the most diverse cell populations in terms of their anatomical location and functional specialization during both homeostasis and disease. Although it has been shown in different fate mapping models that some macrophages present in adult tissues are already established during fetal development, their exact origins are still under debate. In the current study, we developed a fate mapping strain, based on the Kit locus, which allowed us to readdress "the origins" question. Different types of macrophages from various adult tissues were traced to their fetal or adult sources by inducing labeling in precursors at several time points either during fetal development or in adult mice. We show that all adult macrophages, resident or infiltrating, are progenies of classical hematopoietic stem cells (HSC) with the exception of microglia and, partially epidermal Langerhans cells, which are yolk sac (YS)-derived.

Ribonuclease like 5 regulates zebrafish yolk extension by suppressing a p53-dependent DNA damage response pathway.

Ribonuclease like 5 (Rnasel5) is a novel member of the zebrafish ribonuclease A family and its expression is increased during early embryogenesis. However, the in vivo biological function of Rnasel5 remains to be elucidated. Here, we report that knockdown of Rnasel5 by morhpolinos caused shrunken yolk extension as well as increased DNA damage at yolk syncytial layer and external tissue layers via the activation of p53 pathway. In addition, the morphological defects caused by Rnasel5 knockdown can be partially rescued by mRNA injection. Our findings provide the first functional characterization of Rnasel5 in zebrafish development and reveal its critical role in yolk extension by modulation of the p53 pathway.

Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage.

We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens.

Extrinsic and intrinsic control by EKLF (KLF1) within a specialized erythroid niche.

The erythroblastic island provides an important nutritional and survival support niche for efficient erythropoietic differentiation. Island integrity is reliant on adhesive interactions between erythroid and macrophage cells. We show that erythroblastic islands can be formed from single progenitor cells present in differentiating embryoid bodies, and that these correspond to erythro-myeloid progenitors (EMPs) that first appear in the yolk sac of the early developing embryo. Erythroid Krüppel-like factor (EKLF; KLF1), a crucial zinc finger transcription factor, is expressed in the EMPs, and plays an extrinsic role in erythroid maturation by being expressed in the supportive macrophage of the erythroblastic island and regulating relevant genes important for island integrity within these cells. Together with its well-established intrinsic contributions to erythropoiesis, EKLF thus plays a coordinating role between two different cell types whose interaction provides the optimal environment to generate a mature red blood cell.

Conservation of placentation during the tertiary radiation of mammals in South America.

The eutherian placenta is considered to possess great plasticity, but it is not clear how this variation reflects adaptation to different ecological niches. Because South America was isolated for most of the Tertiary, it represents a natural laboratory to examine this question. We here describe placentation in three South American groups: Xenarthra have been part of the fauna from at least the mid-Paleocene whereas caviomorph rodents and Neotropical primates are each derived from a single founder that reached South America in the Eocene and Oligocene, respectively. The common ancestor of Xenarthra had a villous, haemochorial placenta, from which the labyrinthine, endotheliochorial placenta of sloths later evolved. Placentation in Caviomorpha follows an extraordinary stable pattern, characterized by a haemomonochorial, labyrinthine and highly lobed structure with specialized growing areas. This pattern was present before arrival of these rodents in South America and enabled a successful radiation especially during the spread of grasslands. Neotropical primates have haemochorial, trabecular placentas with a specialized maternal blood supply; a pattern that contrasts with that of Old World monkeys and may have been present in the founder generation on arrival in South America. In conclusion, there is a dichotomy within Xenarthra but otherwise the ancient South American mammals do not show much variation in principal placental characters. Thus, the successful radiation of these three groups, and their adaptation to diverse ecological niches, did not require substantial alterations in placentation.

Free blastocyst and implantation stages in the European brown hare: correlation between ultrasound and histological data.

The European brown hare (Lepus europaeus) is the only species with superconception, whereby the maternal reproductive tract hosts two sets of conceptuses at different developmental stages. The embryonic development of the hare has not yet been described. To understand the mechanism of superconception, we studied oviduct transport and implantation stages by embryo flushing and live high-resolution ultrasound. Ultrasound data of implantation stages is correlated with histology. In the oviduct, a mucin coat is deposited on the zona pellucida. The blastocysts enter the uterine horns on Day 5, 1 day later than in the rabbit, and directly expand approximately threefold. Spacing is accompanied by peristaltic movement of the endometrium. The mucin coat disappears and the conceptuses attach. The yolk-sac expands in the blastocoel and syncytial knobs invade the antimesometrial endometrium. Maternal blood lacunae appear in the mesometrial endometrial folds, which are subsequently invaded by the syncytiotrophoblast. The haemochorial chorioallantoic placenta forms. The yolk-sac cavity is gradually replaced by the allantois and finally by the exocoel. The different reproductive strategies of the precocial hare and the altricial rabbit are discussed. We assume that the lagomorph-specific mucin coat and the hare-specific delay of the oviduct-uterine transition are prerequisites for superconception.

GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment.

Recent studies have established that during embryonic development, hematopoietic progenitors and stem cells are generated from hemogenic endothelium precursors through a process termed endothelial to hematopoietic transition (EHT). The transcription factor RUNX1 is essential for this process, but its main downstream effectors remain largely unknown. Here, we report the identification of Gfi1 and Gfi1b as direct targets of RUNX1 and critical regulators of EHT. GFI1 and GFI1B are able to trigger, in the absence of RUNX1, the down-regulation of endothelial markers and the formation of round cells, a morphologic change characteristic of EHT. Conversely, blood progenitors in Gfi1- and Gfi1b-deficient embryos maintain the expression of endothelial genes. Moreover, those cells are not released from the yolk sac and disseminated into embryonic tissues. Taken together, our findings demonstrate a critical and specific role of the GFI1 transcription factors in the first steps of the process leading to the generation of hematopoietic progenitors from hemogenic endothelium.

Whole embryo imaging of hematopoietic cell emergence and migration.

The use of transgenic mice in which tissue or lineage-specific, cell-restricted promoters drive fluorescent reporters has recently been reported as a means to follow the in vivo migration of various hematopoietic cells during murine development. At present there is limited ability of these approaches to image the emergence of the first hematopoietic cell subsets due to lack of unique markers that define those hematopoietic cells. We have utilized whole embryo analysis via immunostaining and confocal laser-scanning microscopic (CLSM) imaging to define the emergence of the first hematopoietic elements in the yolk sac of the developing conceptus. The methods employed to examine yolk sac hematopoiesis may be applied to hematopoietic cell emergence in the embryo proper or fetal liver in the generation of a complete map of hematopoietic ontogeny.

A transient definitive erythroid lineage with unique regulation of the ß-globin locus in the mammalian embryo.

A transient erythromyeloid wave of definitive hematopoietic progenitors (erythroid/myeloid progenitors [EMPs]) emerges in the yolk sac beginning at embryonic day 8.25 (E8.25) and colonizes the liver by E10.5, before adult-repopulating hematopoietic stem cells. At E11.5, we observe all maturational stages of erythroid precursors in the liver and the first definitive erythrocytes in the circulation. These early fetal liver erythroblasts express predominantly adult ß-globins and the definitive erythroid-specific transcriptional modifiers c-myb, Sox6, and Bcl11A. Surprisingly, they also express low levels of "embryonic" ßH1-, but not εy-, globin transcripts. Consistent with these results, RNA polymerase and highly modified histones are found associated with ßH1- and adult globin, but not εy-globin, genes. E11.5 definitive proerythroblasts from mice transgenic for the human ß-globin locus, like human fetal erythroblasts, express predominately human γ-, low ß-, and no ε-globin transcripts. Significantly, E9.5 yolk sac-derived EMPs cultured in vitro have similar murine and human transgenic globin expression patterns. Later liver proerythroblasts express low levels of γ-globin, while adult marrow proerythroblasts express only ß-globin transcripts. We conclude that yolk sac-derived EMPs, the first of 2 origins of definitive erythropoiesis, express a unique pattern of globin genes as they generate the first definitive erythrocytes in the liver of the mammalian embryo.

Alternative Runx1 promoter usage in mouse developmental hematopoiesis.

The interest in stem cell based therapies has emphasized the importance of understanding the cellular and molecular mechanisms by which stem cells are generated in ontogeny and maintained throughout adult life. Hematopoietic stem cells (HSCs) are first found in clusters of hematopoietic cells budding from the luminal wall of the major arteries in the developing mammalian embryo. The transcription factor Runx1 is critical for their generation and is specifically expressed at sites of HSC generation, prior to their formation. To understand better the transcriptional hierarchies that converge on Runx1 during HSC emergence, we have initiated studies into its transcriptional regulation. Here we systematically analyzed Runx1 P1 and P2 alternative promoter usage in hematopoietic sites and in sorted cell populations during mouse hematopoietic development. Our results indicate that Runx1 expression in primitive erythrocytes is largely P2-derived, whilst in definitive hematopoietic stem and/or progenitor cells from the yolk sac or AGM and vitelline and umbilical arteries both the distal P1 and proximal P2 promoters are active. After cells have migrated to the fetal liver, the P1 gradually becomes the main hematopoietic promoter and remains this into adulthood. In addition, we identified a novel P2-derived Runx1 isoform.

A modified yolk biopsy technique improves survivorship of turtle eggs.

Nongenetic maternal contributions, such as steroid hormones, have received much attention in recent years because they have the potential to influence offspring phenotype. Research in oviparous taxa has demonstrated that there is among-species variability in their response to these maternal contributions. However, studies in chelonians and crocodilians have been hampered by the fact that techniques involving egg manipulations that breach the eggshell routinely result in massive egg mortality. In this study, we present an improved yolk manipulation technique that resulted in increased egg survival (in excess of 70% survival) in the red-eared slider turtle (Trachemys scripta) and that may be broadly applicable to other species. By elevating survival to a level on par with other oviparous taxa, this method permits a more thorough exploration of reptilian egg physiology and allows for studies that examine traits in both the egg and the resulting hatchling.