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.

Atypical protein kinase C is essential for embryonic vascular development in mice.

The atypical PKC (aPKC) subfamily constitutes PKCζ and PKCλ in mice, and both aPKC isoforms have been proposed to be involved in regulating various endothelial cell (EC) functions. However, the physiological function of aPKC in ECs during embryonic development has not been well understood. To address this question, we utilized Tie2-Cre to delete PKCλ alone (PKCλ-SKO) or both PKCλ and PKCζ (DKO) in ECs, and found that all DKO mice died at around the embryonic day 11.5 (E11.5), whereas a small proportion of PKCλ-SKO mice survived till birth. PKCλ-SKO embryos also exhibited less phenotypic severity than DKO embryos at E10.5 and E11.5, suggesting a potential compensatory role of PKCζ for PKCλ in embryonic ECs. We then focused on DKO embryos and investigated the effects of aPKC deficiency on embryonic vascular development. At E9.5, deletion of both aPKC isoforms reduced the diameters of vitelline artery and vein, and decreased branching from both vitelline vessels in yolk sac. Ablation of both aPKC isoforms also disrupted embryonic angiogenesis in head and trunk at the same stage, increasing apoptosis of both ECs and non-ECs. Taken together, our results demonstrated that aPKC in ECs plays an essential role in regulating cell apoptosis, angiogenesis, and embryonic survival.

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.

The expression of zebrafish NAD(P)H:quinone oxidoreductase 1(nqo1) in adult organs and embryos.

NQO1, NAD(P)H: quinone oxidoreductase 1, was first identified in rat and its role has been extensively studied. Even the roles of NQO1 in the maintenance of physiological function and disease were largely addressed, whether the tissue specific functions of the NQO1 in organ development remains unknown. In the current study, we identified two NQO1 isoforms (isoform 1 and isoform 2) and examined the expression of nqo1 variants in adult zebrafish organs and embryos at different stages. In adult organs, RT-PCR result indicated that nqo1 variant 1 was mainly expressed in stomach and intestine, while nqo1 variant 2 was expressed in all organs investigated except for heart. Further, RT-PCR result showed that the nqo1 variant 1 and variant 2 were expressed at all the embryonic stages, but nqo1 variant 1 expression level was much lower than that of nqo1 variant 2. To specifically examine the expression pattern of these two different nqo1 variants, we did whole mount in situ hybridization and the results demonstrated that, both of them were maternally expressed at 8-cell stage, and they were all expressed ubiquitously at early stage. At 24 hpf, nqo1 variant 2 was mainly expressed in yolk cells, and slightly in head and eyes. At 48 hpf, nqo1 variant 2 was restricted in lateral line neuromasts. From 72 hpf to 144 hpf, nqo1 variant 2 was mainly restricted in branchial arch, liver, swimming bladder and lateral line neuromasts, while from 124 hpf to 192 hpf, nqo1 variant 2 only restricted in liver, and disappeared in lateral line neuromasts. On the contrary, at the late embryonic stage, nqo1 variant 1 was only expressed in liver and swimming bladder while not in branchial arch and lateral line neuromasts. In conclusion, we systematically analyzed the expression pattern of nqo1 variant 1 and variant 2 in zebrafish at different embryonic stages, and our data implied the possible role of nqo1 in regulating liver, branchial arch and lateral neuromasts development.

Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny.

During embryogenesis, lymphoid tissue inducer (LTi) cells are essential for lymph node organogenesis. These cells are part of the innate lymphoid cell (ILC) family. Although their earliest embryonic hematopoietic origin is unclear, other innate immune cells have been shown to be derived from early hemogenic endothelium in the yolk sac as well as the aorta-gonad-mesonephros. A proper model to discriminate between these locations was unavailable. In this study, using a Cxcr4-CreERT2 lineage tracing model, we identify a major contribution from embryonic hemogenic endothelium, but not the yolk sac, toward LTi progenitors. Conversely, embryonic LTi cells are replaced by hematopoietic stem cell-derived cells in adults. We further show that, in the fetal liver, common lymphoid progenitors differentiate into highly dynamic alpha-lymphoid precursor cells that, at this embryonic stage, preferentially mature into LTi precursors and establish their functional LTi cell identity only after reaching the periphery.

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.

Secreted metalloproteases ADAMTS9 and ADAMTS20 have a non-canonical role in ciliary vesicle growth during ciliogenesis.

Although hundreds of cytosolic or transmembrane molecules form the primary cilium, few secreted molecules are known to contribute to ciliogenesis. Here, homologous secreted metalloproteases ADAMTS9 and ADAMTS20 are identified as ciliogenesis regulators that act intracellularly. Secreted and furin-processed ADAMTS9 bound heparan sulfate and was internalized by LRP1, LRP2 and clathrin-mediated endocytosis to be gathered in Rab11 vesicles with a unique periciliary localization defined by super-resolution microscopy. CRISPR-Cas9 inactivation of ADAMTS9 impaired ciliogenesis in RPE-1 cells, which was restored by catalytically active ADAMTS9 or ADAMTS20 acting in trans, but not by their proteolytically inactive mutants. Their mutagenesis in mice impaired neural and yolk sac ciliogenesis, leading to morphogenetic anomalies resulting from impaired hedgehog signaling, which is transduced by primary cilia. In addition to their cognate extracellular proteolytic activity, ADAMTS9 and ADAMTS20 thus have an additional proteolytic role intracellularly, revealing an unexpected regulatory dimension in ciliogenesis.

Yolk sac-embryo distance in correlation with soluble form of vascular endothelial growth factor levels in pregnancy with potentially reserved evolutivity.

AIMS: Embryonic demise is a frequent complication of the first trimester pregnancy. The purpose of this study was to evaluate the correlation between a serum biomarker, the soluble form of the vasculo-endothelial growth factor (sFlt-1) and the distance between the yolk sac (YS) and embryo (DYSE), determined by ultrasonography. MATERIAL AND METHODS: The study was a prospective case-control study that included 2 groups of patients - the control group with 81 first-trimester pregnancies in evolution and the case group with 89 first-trimester pregnancies with a potentially reserved evolutivity. RESULTS: A correlation between the serum level of sFlt-1 and DYSE in embryos with crown-rump length (CRL) greater than 5 mm was identified, showing that a DYSE ≤3 mm correlates with a low level of sFlt-1 (p<0.05) and a DYSE> 4 mm correlates with an increased level of sFlt-1 (p<0.05). CONCLUSIONS: A low level of sFlt-1 associated with a distance between the embryo and yolk sac of small dimensions, respectively <3 mm, correlates with an increased rate of non-viable embryos. This correlation between an ultrasound and a serum parameter is of great value and brings important information about the viability of firsttrimester pregnancies.

Hemogenic Endothelial Fate Mapping Reveals Dual Developmental Origin of Mast Cells.

Hematopoiesis occurs in distinct waves. "Definitive" hematopoietic stem cells (HSCs) with the potential for all blood lineages emerge in the aorta-gonado-mesonephros, while "primitive" progenitors, whose potential is thought to be limited to erythrocytes, megakaryocytes, and macrophages, arise earlier in the yolk sac (YS). Here, we questioned whether other YS lineages exist that have not been identified, partially owing to limitations of current lineage tracing models. We established the use of Cdh5-CreERT2 for hematopoietic fate mapping, which revealed the YS origin of mast cells (MCs). YS-derived MCs were replaced by definitive MCs, which maintained themselves independently from the bone marrow in the adult. Replacement occurred with tissue-specific kinetics. MCs in the embryonic skin, but not other organs, remained largely YS derived prenatally and were phenotypically and transcriptomically distinct from definite adult MCs. We conclude that within myeloid lineages, dual hematopoietic origin is shared between macrophages and MCs.

Fetal monocytes and the origins of tissue-resident macrophages.

Tissue-resident macrophages have pivotal functions for tissue defense and homeostasis. Two main discoveries have changed our current understanding of macrophage development: Their embryonic origin and their ability to self-renew throughout the lifespan. It is now well accepted that most tissue-resident macrophages are long-lived cells derived from a transient hematopoietic wave of erythro-myeloid progenitors (EMPs) emerging in the yolk sac. At least two distinct pathways derived from EMPs have been implicated in macrophage development. The first one, c-Myb-independent is giving rise to yolk sac macrophages also called primitive macrophages, and bypassing the classical monocytic intermediates. The second requires c-Myb expression and start once EMPs seed the fetal liver where they generate fetal monocytes. Sequentially, primitive macrophages seed every tissue and will ultimately give rise to microglia in the brain, rapidly isolated by the blood brain barrier, while EMP-derived fetal monocytes infiltrate every other tissues and gradually generate the major pool of adult tissue-resident macrophages by diluting the initial primitive macrophage contribution. A third wave of hematopoietic stem cells (HSC)-derived monocytes is also emerging from the fetal liver to contribute to the long-lived macrophage pool established at birth while the adult hematopoiesis is only starting in the bone marrow. We propose here to review recent insights about the different embryonic hematopoietic programs responsible for the generation of long-lived tissue-resident macrophages and their maintenance after birth.

Runx1 is sufficient for blood cell formation from non-hemogenic endothelial cells in vivo only during early embryogenesis.

Hematopoietic cells differentiate during embryogenesis from a population of endothelial cells called hemogenic endothelium (HE) in a process called the endothelial-to-hematopoietic transition (EHT). The transcription factor Runx1 is required for EHT, but for how long and which endothelial cells are competent to respond to Runx1 are not known. Here, we show that the ability of Runx1 to induce EHT in non-hemogenic endothelial cells depends on the anatomical location of the cell and the developmental age of the conceptus. Ectopic expression of Runx1 in non-hemogenic endothelial cells between embryonic day (E) 7.5 and E8.5 promoted the formation of erythro-myeloid progenitors (EMPs) specifically in the yolk sac, the dorsal aorta and the heart. The increase in EMPs was accompanied by a higher frequency of HE cells able to differentiate into EMPs in vitro Expression of Runx1 just 1 day later (E8.5-E9.5) failed to induce the ectopic formation of EMPs. Therefore, endothelial cells, located in specific sites in the conceptus, have a short developmental window of competency during which they can respond to Runx1 and differentiate into blood cells.

Yolk sac macrophage progenitors traffic to the embryo during defined stages of development.

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.

A Yolk Sac Larger Than 5 mm Suggests an Abnormal Fetal Karyotype, Whereas an Absent Embryo Indicates a Normal Fetal Karyotype.

OBJECTIVES: It is very hard to estimate an abnormal or normal fetal karyotype in miscarriage before surgery. We investigated whether the abnormal fetal karyotype in early miscarriage could be estimated by comprehensive ultrasonographic findings by a multivariate analysis. METHODS: One hundred fifty-one patients with early miscarriage (<12 weeks' gestation) were selected in our hospital. The clinical characteristics were compared between pregnant women carrying a fetus with an abnormal karyotype and those with a normal one, and the size and configuration of the gestational sac, yolk sac, and embryo at diagnosis of early miscarriage were also evaluated. RESULTS: The rate of abnormal fetal karyotypes was 66.2 % (100 of 151). A maternal age older than 35 years (odds ratio, 3.2; 95% confidence interval, 1.4-7.4; P = .005), yolk sac larger than 5 mm (odds ratio, 6.2; 95% confidence interval, 2.2-22.7, P < .001), and absent embryo (odds ratio, 0.40; 95% confidence interval, 0.16-0.95; P = .038) were independent markers for predicting an abnormal fetal karyotype by multiple logistic regression analysis. CONCLUSIONS: At the point of early miscarriage diagnosis, a yolk sac larger than 5 mm suggests an abnormal fetal karyotype, whereas an absent embryo indicates a normal fetal karyotype.

Restricted intra-embryonic origin of bona fide hematopoietic stem cells in the chicken.

Hematopoietic stem cells (HSCs), which are responsible for blood cell production, are generated during embryonic development. Human and chicken embryos share features that position the chicken as a reliable and accessible alternative model to study developmental hematopoiesis. However, the existence of HSCs has never been formally proven in chicken embryos. Here, we have established a complete cartography and quantification of hematopoietic cells in the aorta during development. We demonstrate the existence of bona fide HSCs, originating from the chicken embryo aorta (and not the yolk sac, allantois or head), through an in vivo transplantation assay. Embryos transplanted in ovo with GFP embryonic tissues on the chorio-allantoic membrane provided multilineage reconstitution in adulthood. Historically, most breakthrough discoveries in the field of developmental hematopoiesis were first made in birds and later extended to mammals. Our study sheds new light on the avian model as a valuable system to study HSC production and regulation in vivo.

Essential roles for Cdx in murine primitive hematopoiesis.

The Cdx transcription factors play essential roles in primitive hematopoiesis in the zebrafish where they exert their effects, in part, through regulation of hox genes. Defects in hematopoiesis have also been reported in Cdx mutant murine embryonic stem cell models, however, to date no mouse model reflecting the zebrafish Cdx mutant hematopoietic phenotype has been described. This is likely due, in part, to functional redundancy among Cdx members and the early lethality of Cdx2 null mutants. To circumvent these limitations, we used Cre-mediated conditional deletion to assess the impact of concomitant loss of Cdx1 and Cdx2 on murine primitive hematopoiesis. We found that Cdx1/Cdx2 double mutants exhibited defects in primitive hematopoiesis and yolk sac vasculature concomitant with reduced expression of several genes encoding hematopoietic transcription factors including Scl/Tal1. Chromatin immunoprecipitation analysis revealed that Scl was occupied by Cdx2 in vivo, and Cdx mutant hematopoietic yolk sac differentiation defects could be rescued by expression of exogenous Scl. These findings demonstrate critical roles for Cdx members in murine primitive hematopoiesis upstream of Scl.

Insights into blood cell formation from hemogenic endothelium in lesser-known anatomic sites.

BACKGROUND: Hematopoietic stem and progenitor cells (HSPCs) are generated de novo in the embryo in a process termed the endothelial to hematopoietic transition (EHT). EHT is most extensively studied in the yolk sac and dorsal aorta. Recently new sites of hematopoiesis have been described, including the heart, somites, head, and venous plexus of the yolk sac. RESULTS: We examined sites of HSPC formation in well-studied and in less well-known sites by mapping the expression of the key EHT factor Runx1 along with several other markers by means of confocal microscopy. We identified sites of HSPC formation in the head, heart and somites. We also identified sites of HSPC formation in both the arterial and venous plexuses of the yolk sac, and show that progenitors with lymphoid potential are enriched in hematopoietic clusters in close proximity to arteries. Furthermore, we demonstrate that many of the cells in hematopoietic clusters resemble monocytes or granulocytes based on nuclear shape. CONCLUSIONS: We identified sites of HSPC formation in the head, heart, and somites, confirming that embryonic hematopoiesis is less spatially restricted than previously thought. Furthermore, we show that HSPCs in the yolk sac with lymphoid potential are located in closer proximity to arteries than to veins. Developmental Dynamics 245:1011-1028, 2016. © 2016 Wiley Periodicals, Inc.

Endothelial differentiation of canine yolk sac cells transduced with VEGF.

Yolk sac (YS) is the site of blood-cell production where primitive erythroid cells originate and complete their maturation. YS is a source of precursor cells, however its differentiation potential and suitability for cell therapies are not well described. YS can be a cell source when neovascularization is required. This study characterized YS canine cells, transduced with VEGF, to analyze then using Immunocytochemistry, flow cytometry and real time PCR. Immunocytochemistry: positive expression for CD105, PCNA, VEGF and vWF, flow cytometry for CD105, VEGF, PCNA, OCT-4 and RT-qPCR for VEGF, CD31, CD105, PCNA and FLT - 1, indicating that these cells have characteristics of endothelial progenitor and pluripotency. After transduction, the YS cells changed their morphology and showed endothelial-like cells. We suggest, because of their cell surface phenotype as well as their capacity to differentiate into endothelial-like cells, that canine YS represents a source of cells for neovascularization therapies.

The chromatin regulator Brpf1 regulates embryo development and cell proliferation.

With hundreds of chromatin regulators identified in mammals, an emerging issue is how they modulate biological and pathological processes. BRPF1 (bromodomain- and PHD finger-containing protein 1) is a unique chromatin regulator possessing two PHD fingers, one bromodomain and a PWWP domain for recognizing multiple histone modifications. In addition, it binds to the acetyltransferases MOZ, MORF, and HBO1 (also known as KAT6A, KAT6B, and KAT7, respectively) to promote complex formation, restrict substrate specificity, and enhance enzymatic activity. We have recently showed that ablation of the mouse Brpf1 gene causes embryonic lethality at E9.5. Here we present systematic analyses of the mutant animals and demonstrate that the ablation leads to vascular defects in the placenta, yolk sac, and embryo proper, as well as abnormal neural tube closure. At the cellular level, Brpf1 loss inhibits proliferation of embryonic fibroblasts and hematopoietic progenitors. Molecularly, the loss reduces transcription of a ribosomal protein L10 (Rpl10)-like gene and the cell cycle inhibitor p27, and increases expression of the cell-cycle inhibitor p16 and a novel protein homologous to Scp3, a synaptonemal complex protein critical for chromosome association and embryo survival. These results uncover a crucial role of Brpf1 in controlling mouse embryo development and regulating cellular and gene expression programs.

A lineage of diploid platelet-forming cells precedes polyploid megakaryocyte formation in the mouse embryo.

In this study, we test the assumption that the hematopoietic progenitor/colony-forming cells of the embryonic yolk sac (YS), which are endowed with megakaryocytic potential, differentiate into the first platelet-forming cells in vivo. We demonstrate that from embryonic day (E) 8.5 all megakaryocyte (MK) colony-forming cells belong to the conventional hematopoietic progenitor cell (HPC) compartment. Although these cells are indeed capable of generating polyploid MKs, they are not the source of the first platelet-forming cells. We show that proplatelet formation first occurs in a unique and previously unrecognized lineage of diploid platelet-forming cells, which develop within the YS in parallel to HPCs but can be specified in the E8.5 Runx1-null embryo despite the absence of the progenitor cell lineage.

Loss of PiT-1 results in abnormal endocytosis in the yolk sac visceral endoderm.

PiT-1 protein is a transmembrane sodium-dependent phosphate (Pi) transporter. PiT-1 knock out (KO) embryos die from largely unknown causes by embryonic day (E) 12.5. We tested the hypothesis that PiT-1 is required for endocytosis in the embryonic yolk sac (YS) visceral endoderm (VE). Here we present data supporting that PiT-1 KO results in a YS remodeling defect and decreased endocytosis in the YS VE. The remodeling defect is not due to an upstream cardiomyocyte requirement for PiT-1, as SM22αCre-specific KO of PiT-1 in the developing heart and the YS mesodermal layer (ME) does not recapitulate the PiT-1 global KO phenotype. Furthermore, we find that high levels of PiT-1 protein localize to the YS VE apical membrane. Together these data support that PiT-1 is likely required in YS VE. During normal development maternal immunoglobulin (IgG) is endocytosed into YS VE and accumulates in the apical side of the VE in a specialized lysosome termed the apical vacuole (AV). We have identified a reduction in PiT-1 KO VE cell height and a striking loss of IgG accumulation in the PiT-1 KO VE. The endocytosis genes Tfeb, Lamtor2 and Snx2 are increased at the RNA level. Lysotracker Red staining reveals a loss of distinct AVs, and yolk sacs incubated ex vivo with phRODO Green Dextran for Endocytosis demonstrate a functional loss of endocytosis. As yolk sac endocytosis is controlled in part by microautophagy, but expression of LC3 had not been examined, we investigated LC3 expression during yolk sac development and found stage-specific LC3 RNA expression that is predominantly from the YS VE layer at E9.5. Normalized LC3-II protein levels are decreased in the PiT-1 KO YS, supporting a requirement for PiT-1 in autophagy in the YS. Therefore, we propose the novel idea that PiT-1 is central to the regulation of endocytosis and autophagy in the YS VE.