Redefining the ontogeny of hyalocytes as yolk sac-derived tissue-resident macrophages of the vitreous body.

BACKGROUND: The eye is a highly specialized sensory organ which encompasses the retina as a part of the central nervous system, but also non-neural compartments such as the transparent vitreous body ensuring stability of the eye globe and a clear optical axis. Hyalocytes are the tissue-resident macrophages of the vitreous body and are considered to play pivotal roles in health and diseases of the vitreoretinal interface, such as proliferative vitreoretinopathy or diabetic retinopathy. However, in contrast to other ocular macrophages, their embryonic origin as well as the extent to which these myeloid cells might be replenished by circulating monocytes remains elusive. RESULTS: In this study, we combine transgenic reporter mice, embryonic and adult fate mapping approaches as well as parabiosis experiments with multicolor immunofluorescence labeling and confocal laser-scanning microscopy to comprehensively characterize the murine hyalocyte population throughout development and in adulthood. We found that murine hyalocytes express numerous well-known myeloid cell markers, but concomitantly display a distinct immunophenotype that sets them apart from retinal microglia. Embryonic pulse labeling revealed a yolk sac-derived origin of murine hyalocytes, whose precursors seed the developing eye prenatally. Finally, postnatal labeling and parabiosis established the longevity of hyalocytes which rely on Colony Stimulating Factor 1 Receptor (CSF1R) signaling for their maintenance, independent of blood-derived monocytes. CONCLUSION: Our study identifies hyalocytes as long-living progeny of the yolk sac hematopoiesis and highlights their role as integral members of the innate immune system of the eye. As a consequence of their longevity, immunosenescence processes may culminate in hyalocyte dysfunction, thereby contributing to the development of vitreoretinal diseases. Therefore, myeloid cell-targeted therapies that convey their effects through the modification of hyalocyte properties may represent an interesting approach to alleviate the burden imposed by diseases of the vitreoretinal interface.

Modelling post-implantation human development to yolk sac blood emergence.

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 cell atlas reveals multiorgan functions during human early development.

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.

Lineage tracing clarifies the cellular origin of tissue-resident macrophages in the developing heart.

Tissue-resident macrophages play essential functions in the maintenance of tissue homeostasis and repair. Recently, the endocardium has been reported as a de novo hemogenic site for the contribution of hematopoietic cells, including cardiac macrophages, during embryogenesis. These observations challenge the current consensus that hematopoiesis originates from the hemogenic endothelium within the yolk sac and dorsal aorta. Whether the developing endocardium has such a hemogenic potential requires further investigation. Here, we generated new genetic tools to trace endocardial cells and reassessed their potential contribution to hematopoietic cells in the developing heart. Fate-mapping analyses revealed that the endocardium contributed minimally to cardiac macrophages and circulating blood cells. Instead, cardiac macrophages were mainly derived from the endothelium during primitive/transient definitive (yolk sac) and definitive (dorsal aorta) hematopoiesis. Our findings refute the concept of endocardial hematopoiesis, suggesting that the developing endocardium gives rise minimally to hematopoietic cells, including cardiac macrophages.

Modeling human yolk sac hematopoiesis with pluripotent stem cells.

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.

Ezh2 is essential for the generation of functional yolk sac derived erythro-myeloid progenitors.

Yolk sac (YS) hematopoiesis is critical for the survival of the embryo and a major source of tissue-resident macrophages that persist into adulthood. Yet, the transcriptional and epigenetic regulation of YS hematopoiesis remains poorly characterized. Here we report that the epigenetic regulator Ezh2 is essential for YS hematopoiesis but dispensable for subsequent aorta-gonad-mesonephros (AGM) blood development. Loss of EZH2 activity in hemogenic endothelium (HE) leads to the generation of phenotypically intact but functionally deficient erythro-myeloid progenitors (EMPs), while the generation of primitive erythroid cells is not affected. EZH2 activity is critical for the generation of functional EMPs at the onset of the endothelial-to-hematopoietic transition but subsequently dispensable. We identify a lack of Wnt signaling downregulation as the primary reason for the production of non-functional EMPs. Together, our findings demonstrate a critical and stage-specific role of Ezh2 in modulating Wnt signaling during the generation of EMPs from YS HE.

VEGF, FGF2, and BMP4 regulate transitions of mesoderm to endothelium and blood cells in a human model of yolk sac hematopoiesis.

Exogenous growth factors play an important role in mediating hematopoietic differentiation of human pluripotent stem cells. We explored the role of different factors in early human blood cell production using blast colony formation in methylcellulose as a surrogate assay for yolk sac hematopoiesis. A reporter cell line that read out endothelial (SOX17+) and hematopoietic (RUNX1C+) progenitors facilitated the identification of basic fibroblast growth and vascular endothelial growth factor as critical signals for the progression of mesoderm into endothelium. Bone morphogenetic protein 4 was needed for the subsequent generation of blood from hemogenic endothelium, and this was antagonized by Activin A or high concentrations of the WNT agonist CHIR-99021. Manipulations of the Hedgehog pathway or inhibition of Notch signaling reduced blast colony frequency but did not perturb cell differentiation. These data help to define distinct roles for prerequisite growth factors that commit mesoderm to hemogenic endothelium and subsequently allocate cells to blood lineages.

Megakaryocyte production is sustained by direct differentiation from erythromyeloid progenitors in the yolk sac until midgestation.

The extra-embryonic yolk sac contains the first definitive multipotent hematopoietic cells, denominated erythromyeloid progenitors. They originate in situ prior to the emergence of hematopoietic stem cells and give rise to erythroid, monocytes, granulocytes, mast cells and macrophages, the latter in a Myb transcription factor-independent manner. We uncovered here the heterogeneity of yolk sac erythromyeloid progenitors, at the single cell level, and discriminated multipotent from committed progenitors, prior to fetal liver colonization. We identified two temporally distinct megakaryocyte differentiation pathways. The first occurs in the yolk sac, bypasses intermediate bipotent megakaryocyte-erythroid progenitors and, similar to the differentiation of macrophages, is Myb-independent. By contrast, the second originates later, from Myb-dependent bipotent progenitors expressing Csf2rb and colonize the fetal liver, where they give rise to megakaryocytes and to large numbers of erythrocytes. Understanding megakaryocyte development is crucial as they play key functions during vascular development, in particular in separating blood and lymphatic networks.

Erythroid enucleation: a gateway into a "bloody" world.

Erythropoiesis is an intricate process starting in hematopoietic stem cells and leading to the daily production of 200 billion red blood cells (RBCs). Enucleation is a greatly complex and rate-limiting step during terminal maturation of mammalian RBC production involving expulsion of the nucleus from the orthochromatic erythroblasts, resulting in the formation of reticulocytes. The dynamic enucleation process involves many factors ranging from cytoskeletal proteins to transcription factors to microRNAs. Lack of optimum terminal erythroid maturation and enucleation has been an impediment to optimum RBC production ex vivo. Major efforts in the past two decades have exposed some of the mechanisms that govern the enucleation process. This review focuses in detail on mechanisms implicated in enucleation and discusses the future perspectives of this fascinating process.

The T-box transcription factor Eomesodermin governs haemogenic competence of yolk sac mesodermal progenitors.

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.

Optimal combination of anti-inflammatory components from Chinese medicinal formula Liang-Ge-San.

ETHNOPHARMACOLOGICAL RELEVANCE: Liang-Ge-San (LGS), a traditional Chinese medicine (TCM) formula, is usually used in acute inflammatory diseases in China. AIM OF THE STUDY: This study aims to detect the optimal combination of anti-inflammatory components from LGS. MATERIALS AND METHODS: Four mainly representative components (phillyrin, emodin, baicalin, and liquiritin) from LGS were chosen. The optimal combination was investigated by orthogonal design study. Zebrafish inflammation model was established by lipopolysaccharide (LPS)-yolk microinjection, and then the anti-inflammatory activities of different combinations were determined by survival analysis, changes on inflammatory cells infiltration, the MyD88/NF-κB and MAPK pathways and inflammatory cytokines production. RESULTS: The different combinations of bioactive ingredients from LGS significantly protected zebrafish from LPS-induced inflammation, as evidenced by decreased recruitment of macrophages and neutrophils, inhibition of the MyD88/NF-κB and MAPK pathways and down-regulation of TNF-α and IL-6. Among them, the combination group 8 most significantly protected against LPS. The combination of group 8 is: 0.1 µM of emodin, 2 µM of baicalin, 20 µM of phillyrin and 12.5 µM of liquiritin. CONCLUSION: The optimized combination group 8 exerts the most significant anti-inflammatory activity by inhibiting the recruitment of inflammatory cells, activation of the MyD88/NF-κB and MAPK pathways and the secretion of pro-inflammatory cytokines. This present study provides pharmacological evidences for the further development of new modern Chinese drug from LGS to treat acute inflammatory diseases, but indicated the use of zebrafish in the screening of components from formulas.

Cellular Basis of Embryonic Hematopoiesis and Its Implications in Prenatal Erythropoiesis.

Primitive erythrocytes are the first hematopoietic cells observed during ontogeny and are produced specifically in the yolk sac. Primitive erythrocytes express distinct hemoglobins compared with adult erythrocytes and circulate in the blood in the nucleated form. Hematopoietic stem cells produce adult-type (so-called definitive) erythrocytes. However, hematopoietic stem cells do not appear until the late embryonic/early fetal stage. Recent studies have shown that diverse types of hematopoietic progenitors are present in the yolk sac as well as primitive erythroblasts. Multipotent hematopoietic progenitors that arose in the yolk sac before hematopoietic stem cells emerged likely fill the gap between primitive erythropoiesis and hematopoietic stem-cell-originated definitive erythropoiesis and hematopoiesis. In this review, we discuss the cellular origin of primitive erythropoiesis in the yolk sac and definitive hematopoiesis in the fetal liver. We also describe mechanisms for developmental switches that occur during embryonic and fetal erythropoiesis and hematopoiesis, particularly focusing on recent studies performed in mice.

Equine yolk sac: a stem cells source / Saco vitelino equino: una fuente de células madre

SUMMARY: Mesenchymal stem cells are characterized by in vitro high proliferation and multilineage potential maintenance. This study aimed to isolate and characterize equine YS mesenchymal stem cells and compare these with amniotic membranes. The yolk sac (YS) and amniotic membranes (AM) were obtained from 20 pregnant mares with gestational age around 30 days. Cells were cultured in α-MEM supplemented with 15 % FBS, 1 % antibiotic solution, 1 % L-glutamine and 1 % nonessential amino acids. To cell characterization we used cytogenetic analysis, fibroblast colony-forming unit assays, cell growth curves, immunophenotyping, flow cytometry, differentiation assays and teratoma formation. Results: Both cell sources presented fibroblastoid and epithelioid-like format. The YS cells have lower colony formation potential then AM ones, 3 versus 8 colonies per 103 plated cells. However, YS cells grew progressively while AM cells showed steady. Both, the YS and amnion cells immunolabeled for Oct-4, Nanog, SSEA-3, cytokeratin 18, PCNA, and vimentin. In addition, presented mesenchymal, hematopoietic, endothelial and pluripotency markers in flow cytometry. Discussion: Both cell sources presented high plasticity and differed into osteogenic, adipogenic, and chondrogenic lineages, and no tumor formation in nude mice was observed. The results suggest that horse YS may be useful for cell therapy such as amnion-derived cells.

RESUMEN: Las células madre mesenquimales se caracterizan por una alta proliferación in vitro y un mantenimiento potencial de múltiples líneas. Este estudio tuvo como objetivo aislar y caracterizar las células madre mesenquimales del saco vitelino equinas y compararlas con las membranas amnióticas. Se obtuvo el saco vitelino (SV) y las membranas amnióticas (MA) de 20 yeguas preñadas con edad gestacional de aproximadamente 30 días. Las células se cultivaron en α -MEM suplementado con 15 % de FBS, 1 % de solución antibiótica, 1 % de L-glutamina y 1 % de aminoácidos no esenciales. Para la caracterización celular utilizamos análisis citogenéticos, ensayos de unidades de colonias de fibroblastos, curvas de crecimiento celular, inmunofenotipaje, citometría de flujo, ensayos de diferenciación y formación de teratomas. Ambas fuentes celulares presentaron formato fibroblastoideo y epitelioide. Las células SV tienen un potencial de formación de colonias más bajo que las de MA, 3 versus 8 colonias por 103 células en placa. Sin embargo, las células SV crecieron progresivamente mientras que las células MA se mostraron estables. Tanto las células YS como las células amnios están inmunomarcadas para Oct-4, Nanog, SSEA-3, citoqueratina 18, PCNA y vimentina. Además, presentó marcadores mesenquimales, hematopoyéticos, endoteliales y pluripotenciales en citometría de flujo. Ambas fuentes celulares presentaron alta plasticidad y diferían en linajes osteogénicos, adipogénicos y condrogénicos, y no se observó formación de tumores en ratones. Los resultados sugieren que el SV de caballo puede ser útil para la terapia celular, como las células derivadas de amnios.

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

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

Deciphering human macrophage development at single-cell resolution.

Macrophages are the first cells of the nascent immune system to emerge during embryonic development. In mice, embryonic macrophages infiltrate developing organs, where they differentiate symbiotically into tissue-resident macrophages (TRMs)1. However, our understanding of the origins and specialization of macrophages in human embryos is limited. Here we isolated CD45+ haematopoietic cells from human embryos at Carnegie stages 11 to 23 and subjected them to transcriptomic profiling by single-cell RNA sequencing, followed by functional characterization of a population of CD45+CD34+CD44+ yolk sac-derived myeloid-biased progenitors (YSMPs) by single-cell culture. We also mapped macrophage heterogeneity across multiple anatomical sites and identified diverse subsets, including various types of embryonic TRM (in the head, liver, lung and skin). We further traced the specification trajectories of TRMs from either yolk sac-derived primitive macrophages or YSMP-derived embryonic liver monocytes using both transcriptomic and developmental staging information, with a focus on microglia. Finally, we evaluated the molecular similarities between embryonic TRMs and their adult counterparts. Our data represent a comprehensive characterization of the spatiotemporal dynamics of early macrophage development during human embryogenesis, providing a reference for future studies of the development and function of human TRMs.

Yolk-sac-derived macrophages progressively expand in the mouse kidney with age.

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.

Older people are more likely to develop kidney disease, which increases their risk of having other conditions such as a heart attack or stroke and, in some cases, can lead to their death. Older kidneys are less able to repair themselves after an injury, which may help explain why aging contributes to kidney disease. Another possibility is that older kidneys are more susceptible to excessive inflammation. Learning more about the processes that lead to kidney inflammation in older people might lead to better ways to prevent or treat their kidney disease. Immune cells called macrophages help protect the body from injury and disease. They do this by triggering inflammation, which aides healing. Too much inflammation can be harmful though, making macrophages a prime suspect in age-related kidney harm. Studying these immune cells in the kidney and how they change over the lifespan could help scientists to better understand age-related kidney disease. Now, Ide, Yahara et al. show that one type of macrophage is better at multiplying in older kidneys. In the experiments, mice were genetically engineered to make a fluorescent red protein in one kind of macrophage. This allowed Ide, Yahara et al. to track these immune cells as the mice aged. The experiments showed that this subgroup of cells is first produced when the mice are embryos. They stay in the mouse kidneys into adulthood, and are so prolific that, over time, they eventually become the most common macrophage in older kidneys. The fact that one type of embryonically derived macrophage takes over with age may explain the increased inflammation and reduced repair capacity seen in aging kidneys. More studies will help scientists to understand how these particular cells contribute to age-related changes in susceptibility to kidney disease.

Megalin: A Novel Endocytic Receptor for Prorenin and Renin.

Megalin is an endocytic receptor contributing to protein reabsorption. Impaired expression or trafficking of megalin increases urinary renin and allowed the detection of prorenin, which normally is absent in urine. Here, we investigated (pro)renin uptake by megalin, using both conditionally immortalized proximal tubule epithelial cells and Brown Norway Rat yolk sac cells (BN16). To distinguish binding and internalization, cells were incubated with recombinant human (pro)renin at 4°C and 37°C, respectively. (Pro)renin levels were assessed by immunoradiometric assay. At 4°C, BN16 cells bound 3× more prorenin than renin, suggestive for a higher affinity of prorenin. Similarly, at 37°C, prorenin accumulated at 3- to 4-fold higher levels than renin in BN16 cells. Consequently, depletion of medium prorenin (but not renin) content occurred after 24 hours. No such differences were observed in conditionally immortalized proximal tubule epithelial cells, and M6P (mannose-6-phosphate) greatly reduced conditionally immortalized proximal tubule epithelial cells (pro)renin uptake, suggesting that these cells accumulate (pro)renin largely via M6P receptors. M6P did not affect (pro)renin uptake in BN16 cells. Yet, inhibiting megalin expression with siRNA greatly reduced (pro)renin binding and internalization by BN16 cells. Furthermore, treating BN16 cells with albumin, an endogenous ligand of megalin, also decreased binding and internalization of (pro)renin, while deleting the (pro)renin receptor affected the latter only. Exposing prorenin's prosegment with the renin inhibitor aliskiren dramatically increased prorenin binding, while after prosegment cleavage with trypsin prorenin binding was identical to that of renin. In conclusion, megalin might function as an endocytic receptor for (pro)renin and displays a preference for prorenin. Megalin-mediated endocytosis requires the (pro)renin receptor.

Fetal monocytes possess increased metabolic capacity and replace primitive macrophages in tissue macrophage development.

Tissue-resident macrophages (MΦTR ) originate from at least two distinct waves of erythro-myeloid progenitors (EMP) arising in the yolk sac (YS) at E7.5 and E8.5 with the latter going through a liver monocyte intermediate. The relative potential of these precursors in determining development and functional capacity of MΦTR remains unclear. Here, we studied development of alveolar macrophages (AM) after single and competitive transplantation of different precursors from YS, fetal liver, and fetal lung into neonatal Csf2ra-/- mice, which lack endogenous AM. Fetal monocytes, promoted by Myb, outcompeted primitive MΦ (pMΦ) in empty AM niches and preferentially developed to mature AM, which is associated with enhanced mitochondrial respiratory and glycolytic capacity and repression of the transcription factors c-Maf and MafB. Interestingly, AM derived from pMΦ failed to efficiently clear alveolar proteinosis and protect from fatal lung failure following influenza virus infection. Thus, our data demonstrate superior developmental and functional capacity of fetal monocytes over pMΦ in AM development and underlying mechanisms explaining replacement of pMΦ in fetal tissues.

In vivo generation of haematopoietic stem/progenitor cells from bone marrow-derived haemogenic endothelium.

It is well established that haematopoietic stem and progenitor cells (HSPCs) are generated from a transient subset of specialized endothelial cells termed haemogenic, present in the yolk sac, placenta and aorta, through an endothelial-to-haematopoietic transition (EHT). HSPC generation via EHT is thought to be restricted to the early stages of development. By using experimental embryology and genetic approaches in birds and mice, respectively, we document here the discovery of a bone marrow haemogenic endothelium in the late fetus/young adult. These cells are capable of de novo producing a cohort of HSPCs in situ that harbour a very specific molecular signature close to that of aortic endothelial cells undergoing EHT or their immediate progenies, i.e., recently emerged HSPCs. Taken together, our results reveal that HSPCs can be generated de novo past embryonic stages. Understanding the molecular events controlling this production will be critical for devising innovative therapies.

Heat shock protein 60 regulates yolk sac erythropoiesis in mice.

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.