Early Yolk Sac Research Articles

Characterization of gene regulatory networks underlying key properties in human hematopoietic stem cell ontogeny.

Human hematopoiesis starts at early yolk sac and undergoes site- and stage-specific changes over development. The intrinsic mechanism underlying property changes in hematopoiesis ontogeny remains poorly understood. Here, we analyzed single-cell transcriptome of human primary hematopoietic stem/progenitor cells (HSPCs) at different developmental stages, including yolk-sac (YS), AGM, fetal liver (FL), umbilical cord blood (UCB) and adult peripheral blood (PB) mobilized HSPCs. These stage-specific HSPCs display differential intrinsic properties, such as metabolism, self-renewal, differentiating potentialities etc. We then generated highly co-related gene regulatory network (GRNs) modules underlying the differential HSC key properties. Particularly, we identified GRNs and key regulators controlling lymphoid potentiality, self-renewal as well as aerobic respiration in human HSCs. Introducing selected regulators promotes key HSC functions in HSPCs derived from human pluripotent stem cells. Therefore, GRNs underlying key intrinsic properties of human HSCs provide a valuable guide to generate fully functional HSCs in vitro.

Physiology and diseases of tissue-resident macrophages.

Embryo-derived tissue-resident macrophages are the first representatives of the haematopoietic lineage to emerge in metazoans. In mammals, resident macrophages originate from early yolk sac progenitors and are specified into tissue-specific subsets during organogenesis-establishing stable spatial and functional relationships with specialized tissue cells-and persist in adults. Resident macrophages are an integral part of tissues together with specialized cells: for instance, microglia reside with neurons in brain, osteoclasts reside with osteoblasts in bone, and fat-associated macrophages reside with white adipocytes in adipose tissue. This ancillary cell type, which is developmentally and functionally distinct from haematopoietic stem cell and monocyte-derived macrophages, senses and integrates local and systemic information to provide specialized tissue cells with the growth factors, nutrient recycling and waste removal that are critical for tissue growth, homeostasis and repair. Resident macrophages contribute to organogenesis, promote tissue regeneration following damage and contribute to tissue metabolism and defence against infectious disease. A correlate is that genetic or environment-driven resident macrophage dysfunction is a cause of degenerative, metabolic and possibly inflammatory and tumoural diseases. In this Review, we aim to provide a conceptual outline of our current understanding of macrophage physiology and its importance in human diseases, which may inform and serve the design of future studies.

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.

ONTOGENY IS A CRITICAL DETERMINANT OF NATURAL KILLER CELL POTENTIAL AND FUNCTION

The derivation of natural killer (NK) cells from human pluripotent stem cells (hPSCs) is an exciting alternative for adoptive immunotherapy strategies in the treatment of cancer. While NK cells are component of the embryonic hematopoietic repertoire, their ontogenic origins remain unclear. During mammalian embryogenesis, hematopoietic development in the early yolk sac consists of the (HSC)-independent primitive and erythro-myeloid progenitor (EMP) hematopoietic programs. Neither population is known to harbor lymphoid potential. As we previously observed NK cell development from extra-embryonic-like progenitors derived from hPSCs, we asked whether a yolk sac-derived program possesses NK cell potential. Surprisingly, we determined that EMPs isolated from the yolk sacs of E9.5 mouse embryos harbor robust NK cell potential. Further, these NK cells exhibited a potent degranulation response following stimulation, making them functionally distinct from HSC-derived NK cells. In parallel studies of hPSCs differentiated as previously described, we found that extra-embryonic-like CD34+ myeloid progenitors, similar to murine EMPs, give rise to NK cells that are biased for cytolytic degranulation. In contrast, hPSC-derived CD34+ lymphoid progenitors give rise to NK cells that are phenotypically similar to those derived from cord blood, since both exhibit a poor degranulation response but robustly produce inflammatory cytokines. Collectively, our studies in mouse embryos and human PSCs point to a novel, hematopoietic stem cell (HSC)-independent origin of embryonic NK cells that differ functionally from HSC-derived NK cells. These findings not only identify a myeloid origin to embryonic NK cells, but also have important implications for the design of hPSC-derived NK cell-based therapeutics.

Long-Term Engraftment of ESC-Derived B-1 Progenitor Cells Supports HSC-Independent Lymphopoiesis.

SummaryIt is generally considered that mouse embryonic stem cell (ESC) differentiation into blood cells in vitro recapitulates yolk sac (YS) hematopoiesis. As such, similar to YS-derived B-progenitors, we demonstrate here that ESC-derived B-progenitors differentiate into B-1 and marginal zone B cells, but not B-2 cells in immunodeficient mice after transplantation. ESC-derived B-1 cells were maintained in the recipients for more than 6 months, secreting natural IgM antibodies in vivo. Gene expression profiling displayed a close relationship between ESC- and YS-derived B-1 progenitors. Because there are no hematopoietic stem cells (HSCs) detectable in our ESC differentiation culture, successful long-term engraftment of ESC-derived functional B-1 cells supports the presence of HSC-independent B-1 cell development.

Epidermal γδ T cells originate from yolk sac hematopoiesis and clonally self-renew in the adult.

The murine epidermis harbors two immune cell lineages, Langerhans cells (LCs) and γδ T cells known as dendritic epidermal T cells (DETCs). LCs develop from both early yolk sac (YS) progenitors and fetal liver monocytes before locally self-renewing in the adult. For DETCs, the mechanisms of homeostatic maintenance and their hematopoietic origin are largely unknown. Here, we exploited multicolor fate mapping systems to reveal that DETCs slowly turn over at steady state. Like for LCs, homeostatic maintenance of DETCs is achieved by clonal expansion of tissue-resident cells assembled in proliferative units. The same mechanism, albeit accelerated, facilitates DETC replenishment upon injury. Hematopoietic lineage tracing uncovered that DETCs are established independently of definitive hematopoietic stem cells and instead originate from YS hematopoiesis, again reminiscent of LCs. DETCs thus resemble LCs concerning their maintenance, replenishment mechanisms, and hematopoietic development, suggesting that the epidermal microenvironment exerts a lineage-independent influence on the initial seeding and homeostatic maintenance of its resident immune cells.

Mouse Yolk Sac Hematopoiesis.

The yolk sac is the first observed site of hematopoiesis during mouse ontogeny. Primitive erythroid cells are the most well-recognized cell lineages produced from this tissue. In addition to primitive erythroid cells, several types of hematopoietic cells are present, including multipotent hematopoietic progenitors. Yolk sac-derived blood cells constitute a transient wave of embryonic and fetal hematopoiesis. However, recent studies have demonstrated that some macrophage and B cell lineages derived from the early yolk sac may persist to adulthood. This review discusses the cellular basis of mouse yolk sac hematopoiesis and its contributions to embryonic and adult hematopoietic systems.

Repression of Primitive Erythroid Program Is Critical for the Initiation of Multi-Lineage Hematopoiesis in Mouse Development.

Formation of the hematopoietic cells occurs in multiple steps. The first hematopoietic cells observed during ontogeny are primitive erythrocytes, which are produced in the early yolk sac within a limited temporal window. Multi-lineage hematopoiesis, which supplies almost the entire repertoire of blood cell lineages, lags behind primitive erythropoiesis in the tissue. However, molecular mechanisms regulating sequential generation of primitive erythrocytes and multipotent hematopoietic progenitors in the yolk sac are largely unknown. In this study, the transcription factors involved in the development of hematopoietic cells were examined in purified progenitor cell populations from pluripotent stem cell cultures and from the yolk sac of developing embryos. We found that the earliest committed hematopoietic progenitors highly expressed Gata1, Scl/tal1, and Klf1 genes. Expression of these transcription factors, which is known to form a core erythroid transcriptional network, explained the prompt generation of primitive erythrocytes from these earliest progenitors. Importantly, the multipotent hematopoietic cells, which lack the differentiation potential into primitive erythroid cells, down-regulated these genes during a transition from the earliest committed progenitors. In addition, we showed that Pu.1 is involved in the multipotent cell differentiation through the suppression of erythroid transcription program. We propose that these molecular mechanisms governed by transcription factors form sequential waves of primitive erythropoiesis and multi-lineage hematopoiesis in the early yolk sac of developing embryos. J. Cell. Physiol. 232: 323-330, 2017. © 2016 Wiley Periodicals, Inc.

Abstract 162: Resident Aortic Intimal Mononuclear Phagocytes (MNPs) in the Promotion of Atherosclerosis

Atherosclerosis is an underlying cause of cardiovascular disease (CVD) and a leading cause of morbidity and mortality worldwide. Atherosclerosis promotes CVD through plaque formation, restricted blood flow, and thrombotic events. Macrophage accumulation in plaques, their uptake of cholesterol, and subsequent local death drive disease progression, however, there is growing appreciation for more diverse roles of myeloid cells during disease progression. Here, we focused on characterization of so-called vascular dendritic cells (DCs), which we refer to as aortic mononuclear phagocytes (MNP), that reside under the endothelium in plaque-prone areas. Using en face whole-mount confocal microscopy of aortas, we confirm a uniform resident CD11c+ CX3CR1+ MHCII+ MNP population, even in C57/BL6 mice resistant to atherosclerosis. We find aortic MNPs require M-CSF and Flt3 signaling for survival, but are independent of CCR2 and GM-CSF receptor signaling, making them a distinct myeloid population. They express macrophage-restricted genes LysM and CD64, but not dendritic cell specific genes zBTB46 or L-myc. Lineage-tracing analysis using CD115creER and Flt3cre reporter mice indicate that aortic MNPs likely differentiate from definitive hematopoiesis and not early yolk sac progenitors. Parabiosis experiments show that aortic MNPs are self-maintained independent of blood-born progenitors, failing to exchange with blood progenitors for up to 5 months. Aortic MNPs are different from either typical DCs or macrophages and instead appear to blend distinguishing features of each, more closely resembling macrophages. Using our characterization data, we hope to develop an inducible cell-tracking and a cell-depletion model to differentiate the function of aortic MNPs during the progression of atherosclerosis. Overall, we anticipate these functional analyses will reveal, for the first time, the role of resident aortic MNPs in atherosclerosis.

Distinct Sources of Hematopoietic Progenitors Emerge before HSCs and Provide Functional Blood Cells in the Mammalian Embryo.

Hematopoietic potential arises in mammalian embryos before adult-repopulating hematopoietic stem cells (HSCs). At embryonic day 9.5 (E9.5), we show the first murine definitive erythro-myeloid progenitors (EMPs) have an immunophenotype distinct from primitive hematopoietic progenitors, maturing megakaryocytes and macrophages, and rare B cell potential. EMPs emerge in the yolk sac with erythroid and broad myeloid, but not lymphoid, potential. EMPs migrate to the fetal liver and rapidly differentiate, including production of circulating neutrophils by E11.5. Although the surface markers, transcription factors, and lineage potential associated with EMPs overlap with those found in adult definitive hematopoiesis, they are present in unique combinations or proportions that result in a specialized definitive embryonic progenitor. Furthermore, we find that embryonic stem cell (ESC)-derived hematopoiesis recapitulates early yolk sac hematopoiesis, including primitive, EMP, and rare B cell potential. EMPs do not have long-term potential when transplanted in immunocompromised adults, but they can provide transient adult-like RBC reconstitution.

Are "resting" microglia more "m2"?

OPINION article Front. Immunol., 18 November 2014Sec. Molecular Innate Immunity Volume 5 - 2014 | https://doi.org/10.3389/fimmu.2014.00594

Clonal analysis identifies hemogenic endothelium as the source of the blood-endothelial common lineage in the mouse embryo

AbstractThe first blood and endothelial cells of amniote embryos appear in close association in the blood islands of the yolk sac (YS). This association and in vitro lineage analyses have suggested a common origin from mesodermal precursors called hemangioblasts, specified in the primitive streak during gastrulation. Fate mapping and chimera studies, however, failed to provide strong evidence for a common origin in the early mouse YS. Additional in vitro studies suggest instead that mesodermal precursors first generate hemogenic endothelium, which then generate blood cells in a linear sequence. We conducted an in vivo clonal analysis to determine the potential of individual cells in the mouse epiblast, primitive streak, and early YS. We found that early YS blood and endothelial lineages mostly derive from independent epiblast populations, specified before gastrulation. Additionally, a subpopulation of the YS endothelium has hemogenic activity and displays characteristics similar to those found later in the embryonic hemogenic endothelium. Our results show that the earliest blood and endothelial cell populations in the mouse embryo are specified independently, and that hemogenic endothelium first appears in the YS and produces blood precursors with markers related to definitive hematopoiesis.

Microglia – the brain’s busy bees

Recent years have seen significant changes in the way scientists view microglia and their role in health and disease. For decades, it was presumed that microglia were stationary, inactive immune cells in the brain, waiting for an immunologic insult to call them into action. In contrast, modern imaging techniques have revealed that microglia are constantly in motion, surveying their environment. Lineage tracing studies have led to the understanding that microglia are part of a larger family of cells, called tissue-resident macrophages, which arise from early yolk sac progenitors during embryogenesis and engraft nearly every organ in the body. Microglia, and all tissue-resident macrophages, rely on signaling through CD115 (the colony stimulating factor 1 receptor) for survival, primarily through the ligand, macrophage colony-stimulating factor. However, it is now understood that some microglia have a specific need for another CD115 ligand, Interleukin-34, which is only shared with Langerhans cells in the skin. In contrast to classical views, recent evidence suggests that the primary functions of microglia may occur during postnatal neurodevelopment and adult homeostasis, as absence or impairment of microglia results in a pathology separate from inflammatory immune function. In summary, these advances suggest that microglia might eventually be utilized or targeted to improve disease outcomes via encouraging or enhancing their health-promoting homeostatic functions.

Understanding the role of the microenvironment during definitive hemopoietic development

Hemopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Recent studies in adult bone marrow have identified osteoblasts and endothelial cells as two important supportive cell types within the niche and demonstrated that interactions between HSCs and cellular and extracellular components within the endosteal and perivascular regions are critical for HSC regulation. However, the understanding of the role of the microenvironment in definitive HSC establishment, expansion, and maintenance during embryonic development is extremely limited. This review focuses on what is known about the components of each HSC microenvironment at various developmental stages and their known functional roles.

Modeling Congenital Dyserythropoietic Anemia Type I Through Patient-Derived Induced Pluripotent Stem Cells

AbstractAbstract 3196Congenital dyserythropoietic anemias (CDAs) are rare inherited disorders characterized by impaired red blood cell formation (dyserythropoiesis) and signature cytopathologies. CDA type I is an autosomal recessive disease with macrocytic anemia and occasional bone abnormalities. Erythroid precursors exhibit pathognomonic abnormalities including internuclear chromatin bridges and spongy (“Swiss cheese”) heterochromatin. The disease is caused by biallelic mutations in the gene CDANI (Dgany et al., 2002), which encodes codanin-1, a ubiquitously expressed protein that is believed to have fundamental roles in cell cycle control and chromatin structure (Noy-Lotan et. al, 2009). Animal models for the study of CDA I are suboptimal and clinical samples are scarce. Thus, we have developed an experimental model for the study of CDA I by generating induced pluripotent stem cells (iPSCs) from affected patients. We reprogrammed fibroblasts from CDA I patients and normal subjects using a single lentiviral vector encoding OCT4, KLF4, SOX2, and MYC. The resultant iPSCs exhibited standard criteria for pluripotency and the integrated reprogramming vector was excised using Cre-lox technology.We differentiated CDA I and control iPSCs into erythroid progenitors by inducing the formation of embryoid bodies (EBs) with stepwise additions of supportive cytokines. Beginning at about day 8, hematopoietic progenitors with erythroid potential were detected within EBs and as free-floating cells in the medium. Our differentiation protocol showed two waves of erythroid precursor production. Early EBs (days 12 to 23) produced erythroid cells that expressed mainly epsilon globin, resembling early yolk sac type “primitive” erythropoiesis. In contrast, erythroblasts produced from later EBs (days 27 to 50) expressed mainly gamma globins, resembling “definitive” erythroid cells produced by late stage yolk sac and fetal liver. Our preliminary studies, indicate that CDA I iPSCs produce normal numbers of primitive and definitive erythrocytes. No defects in survival or maturation were detected by flow cytometry assessing the expression of annexin V and the developmental stage markers CD235/CD71/forward scatter. However, definitive type (but not primitive) erythroblasts derived from CDA I iPSCs exhibit some characteristic pathological features including occasional internuclear chromatin bridging visible by light microscopy and spongy “Swiss cheese” heterochromatin revealed by transmission electron microscopy. Thus, patient-derived iPSCs can model at least some aspects of CDA I and provide the basis for future studies to define the actions of codanin-1 and the pathophysiology of this disorder. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Development of yolk sac inversion in Galea spixii and Cavia porcellus (Rodentia, Caviidae)

Caviomorph development includes an inverted yolk sac. Since principle processes are not understood, we investigated its differentiation in Galea and re-examined material from the guinea pig. Galea showed the typical caviomorph conditions in blastocyst development and the nature of the definitive yolk sac, formed of the visceral layer that became villous, proliferative, vascularized and attached to the uterus and placenta. In contrast to what was known before, in both species parts of the parietal yolk sac and a yolk sac cavity were temporarily present. Data suggest that early yolk sac development in caviomorphs is more complex than thought before.

Abstract 16786: Er71 Transcriptionally Activates Lmo2 and Regulates Endothelial Development

During early embryonic development, mesodermal progenitor cells give rise to multiple lineages including the cardiovascular, endothelial and hematopoietic lineages. Studies suggest that the extraembryonic yolk sac is a progenitor source for endothelial and hematopoietic lineages during embryogenesis. We have previously shown that Ets related protein 71(ER71) regulates the development of endothelial and hematopoietic lineages in the early yolk sac and embryo proper utilizing an ER71 gene knock out mouse model, an ER71 promoter driven EYFP-expressing transgenic mouse model and an doxycycline-inducible ER71-overexpressing ES/EB model. To decipher the ER71 mediated mechanism for lineage specification, we evaluated candidate downstream targets of ER71 using qPCR, transcriptome analysis, ChIP and transcriptional assays in the ER71 global gene knockout mouse and doxycycline inducible ER71 overexpressing ES/EB models. Using qPCR, we observed an absence of hematopoietic and endothelial restricted genes in the ER71 mutant yolk sacs and embryo proper compared to their wildtype littermates. We further observed a significant downregulation of Lmo2 in the mutant embryo compared to the wildtype control. Moreover, forced overexpression of ER71 using an inducible ES/EB system revealed that Lmo2 was significantly upregulated within 12 hours following ER71 induction. Analysis of approximately 100kb of the Lmo2 gene revealed multiple potential Ets binding motifs and evaluation of each of these sites using ChIP assays focused our efforts on the 1 kb region in the first intron of the Lmo2 gene. Using an array of biochemical techniques including EMSA, transcriptional assays and mutagenesis our data collectively support the hypothesis that Lmo2 is a direct downstream target of ER71. In summary, the present studies define an ER71-Lmo2 transcriptional network that governs endothelial specification and differentiation during cardiovascular development.

A Systems Biology Approach to Study the Acquisition of Adult Repopulating Potential During Hematopoietic Stem Cell Ontogeny.

Abstract 1479Poster Board I-502Hematopoietic progenitors and stem cells emerge at distinct times and anatomical locations during ontogeny. The first definitive hematopoietic progenitors appear in the yolk sac (YS) around day 8.5 of development (E8.5) while the first cells to meet the functional criteria of hematopoietic stem cell (HSC; cells capable of life-long restoration of the hematopoietic compartment of a recipient whose own hematopoietic system has been destroyed by chemicals or irradiation) can be detected in the aorta-gonads-mesonephros (AGM) at E10 of development. HSC can later be detected in large numbers in the E12.5 placenta, E14.5 fetal liver (FL) and adult bone marrow (BM). Induction of ectopic Cdx4 and HoxB4 expression during murine embryonic stem cell (ESC) differentiation followed by OP9 co-culture yields cells with in vivo hematopoietic repopulating potential (ESC-HSC). We have recently defined the phenotype of ESC-HSC relative to that of HSCs that arise in the embryo during development. Using cell fractionation followed by transplantation into irradiated Rag-2-/-γc-/- mice, we found that ESC-HSC display cell surface markers representative of both embryonic and adult HSC compartments: they express high levels of CD41, are heterogeneous for CD45, express CD150, and lack CD34 expression. We also found that CD150 is a developmentally regulated molecule on the surface of HSC, absent on the earliest HSC compartments of the AGM and placenta, but present on FL and bone marrow HSC. Although hematopoietic cells of the early YS do not display HSC potential when transplanted into the periphery of adult recipients, these cells can acquire this potential if infected with the homeobox gene HoxB4 and cultured on OP9 stroma, similarly to ESC. These data suggest that activation of specific molecular pathways can induce adult repopulating potential. To identify the critical genetic networks regulating the acquisition of adult hematopoietic repopulating potential, we are exploiting prospective purification via flow cytometry and microarray technology to assess the global gene expression profiles of HSC and progenitors from throughout murine ontogeny and embryonic stem cell differentiation. By comparing populations that lack adult repopulating potential (c-kit+CD41+CD34+ E9 YS cells, c-kit+CD41+CD45- ESC-derived cells and VE-cadherin+CD41- ESC-derived cells) with those that possess this activity (VE-cadherin+CD45+ E11.5 AGM, c-kit+CD34medCD45+ E12.5 placenta, Lin-c-kit+Sca-1+CD150+CD48- E14.5 FL, Lin-c-kit+Sca-1+CD150+CD34- BM, and CD41highCD45-CD34- cells from HoxB4 infected ESC expanded on OP9 stroma) we can use a novel gene network inference algorithm dubbed “mode-of-action by network identification” (MNI) to elucidate the key mediators for this functionality. The MNI algorithm predicts the key mediators under a particular perturbation by using a microarray compendium to infer a gene regulatory network that assesses how gene transcripts change in relation to one another and the net external influence. A large compendium with diverse experimental scenarios helps MNI to reverse engineer a comprehensive network model. We are currently using around 11,000 murine gene expression data sets acquired using 430 2.0 Affymetrix array chips to infer a regulatory network model, through which our HSC and progenitors microarray data will be filtered to identify the mode of action of the specific profiles accordingly. By comparing repopulating and non-repopulating populations from mouse ontogeny and ESC, we hope to identify the critical mediators and gene networks regulating the acquisition of adult repopulating potential during development. Disclosures:Daley: iPierian: Consultancy, Equity Ownership; Epizyme: Consultancy; Solasia: Consultancy; MPM Capital: Consultancy.

The influence of different salinity conditions on egg buoyancy and development and yolk sac larval survival and morphometric traits of Baltic Sea sprat (<i>Sprattus sprattus balticus</i> Schneider)

The small pelagic sprat (Sprattus sprattus) is a key ecologic player in the Baltic Sea. However, there is long-term variability in recruitment which is thought to be influenced by fluctuations in abiotic and biotic conditions experienced during the early life stages. This study concentrates on the influence of different ambient salinities on sprat egg development, egg buoyancy and survival as well as early yolk sac larval morphometric traits. Egg buoyancy significantly decreased with increasing salinity experienced during fertilization and/or incubation experiments. Field egg buoyancy measurements in 2007 and 2008 exhibited annual and seasonal differences in specific gravity, potentially associated with changes in adult sprat vertical distribution. Neither egg development time nor the duration of the yolk sac phase differed among salinity treatments. At eye pigmentation, larval standard length exhibited high variance among individuals but did not differ among treatments. The largest ecological impact of salinity experienced during spawning was the modification the buoyancy of eggs and yolk sac larvae, which determines their vertical habitat in the Baltic Sea. There are strong thermo- and oxyclines in the Baltic Sea, and thus salinity can indirectly impact

Expression of AA4.1 marks lymphohematopoietic progenitors in early mouse development

The hematopoietic system of mice is established during the early to midgestational stage of development. However, the earliest lymphohematopoietic progenitors that appear during mouse development have been less well characterized compared with the hematopoietic stem cell compartment of fetal liver and bone marrow. We isolated the earliest lymphohematopoietic progenitors by using embryonic stem (ES) cell culture in vitro. Cells with the c-Kit(+)Lin(-) cell surface phenotype were present abundantly in ES cells cocultured with stromal cell lines. We further separated the cells into two distinct cell subsets based on AA4.1 expression. Although AA4.1(+) and AA4.1(-) cells had equivalent potency to generate myeloid cell lineages, the lymphoid potential in ES-cell-derived cells was largely restricted to the cells expressing AA4.1. The same cell type was present abundantly in the early yolk sac and in fewer numbers (approximately 5% of that in the yolk sac) in the caudal half of the developing embryos. These data suggest that AA4.1 is a cell surface marker that can identify the earliest lymphohematopoietic progenitors in mouse development.