Yolk Sac Cells Research Articles

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.

Abstract 097: Megalin: A Novel Endocytic Receptor for Prorenin and Renin?

Disturbed renal handling of prorenin and renin [(pro)renin] may result in hypertension and renal damage. Megalin is a major endocytic receptor playing an essential role in nutrient reabsorption. Genetic mutations resulting in impaired expression or trafficking of megalin in patients with Dent’s disease or Lowe syndrome caused a 20-40-fold rise of urinary renin levels. Interestingly, the urine of such patients also contained prorenin, which normally is undetectable in urine. Moreover, inhibiting megalin in rodents massively upregulated urinary renin levels. The aim of this study was to investigate (pro)renin binding and internalization by megalin. Immortalized Brown Norway Rat yolk sac cells (BN16) were used, since they abundantly express megalin. To distinguish binding and internalization, cells were incubated at 4°C and 37°C, respectively, with 1000 U/L recombinant human (pro)renin for up to 24 hours. (Pro)renin levels were assessed using an immunoradiometric assay. At 4°C, BN16 cells bound 3 times more prorenin than renin (14184.0±2626.0 vs. 4795.0±986.5 pg/mg of protein, p=0.0155), suggestive for a higher affinity of prorenin. Similarly, at 37°C, prorenin accumulated at 3-fold higher levels than renin in BN16 cells. Consequently, depletion of medium prorenin (but not renin) content occurred after 24 hours. Mannose-6-phosphate (M6P), an antagonist of the (pro)renin-binding M6P receptor, did not affect cellular (pro)renin uptake. Yet, inhibiting megalin expression using siRNAs reduced (pro)renin binding and internalization by >50%. Furthermore, treating cells with albumin, an endogenous ligand of megalin, also decreased binding and internalization of (pro)renin. 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 is a novel endocytic receptor for (pro)renin which determines urinary (pro)renin levels. Megalin displays selectivity towards prorenin in a prosegment-dependent manner, and this explains why urine normally does not contain prorenin, except under conditions where megalin trafficking is disturbed.

Secondary Placental Defects in Cxadr Mutant Mice.

The Coxsackie virus and adenovirus receptor (CXADR) is an adhesion molecule known for its role in virus-cell interactions, epithelial integrity, and organogenesis. Loss of Cxadr causes numerous embryonic defects in mice, notably abnormal development of the cardiovascular system, and embryonic lethality. While CXADR expression has been reported in the placenta, the precise cellular localization and function within this tissue are unknown. Since impairments in placental development and function can cause secondary cardiovascular abnormalities, a phenomenon referred to as the placenta-heart axis, it is possible placental phenotypes in Cxadr mutant embryos may underlie the reported cardiovascular defects and embryonic lethality. In the current study, we determine the cellular localization of placental Cxadr expression and whether there are placental abnormalities in the absence of Cxadr. In the placenta, CXADR is expressed specifically by trophoblast labyrinth progenitors as well as cells of the visceral yolk sac (YS). In the absence of Cxadr, we observed altered expression of angiogenic factors coupled with poor expansion of trophoblast and fetal endothelial cell subpopulations, plus diminished placental transport. Unexpectedly, preserving endogenous trophoblast Cxadr expression revealed the placental defects to be secondary to primary embryonic and/or YS phenotypes. Moreover, further tissue-restricted deletions of Cxadr suggest that the secondary placental defects are likely influenced by embryonic lineages such as the fetal endothelium or those within the extraembryonic YS vascular plexus.

Evaluation of the Therapeutic Potential of Canine Yolk Sac Cells in Dogs with Hips Dysplasia

Daniel Tonin Benedetti1,3, Daniela Amstalden2, Nathia Nathaly Rigoglio1, Danilo Custódio Marques*1, Maria Angelica Miglino1 and Paula Fratini1 Author Affiliations 1Department of Surgery, School of Veterinary Medicine and Animal Science, Brazil 2Rusvet Image Diagnostic Center, Brazil 3VetPrado Veterinary Hospital, Brazil Received: March 14, 2019 | Published: March 21, 2019 Corresponding author: Maria Angelica Miglino, Department of Surgery, School of Veterinary Medicine and Animal Science, Brazil DOI: 10.26717/BJSTR.2019.16.002827

Microglia in Alzheimer's Disease: Risk Factors and Inflammation.

Microglia are resident immune cells in the central nervous system (CNS) that originate from myeloid progenitor cells in the embryonic yolk sac and are maintained independently of circulating monocytes throughout life. In the healthy state, microglia are highly dynamic and control the environment by rapidly extending and retracting their processes. When the CNS is inflamed, microglia can give rise to macrophages, but the regulatory mechanisms underlying this process have not been fully elucidated. Recent genetic studies have suggested that microglial function is compromised in Alzheimer's disease (AD), and that environmental factors such as diet and brain injury also affect microglial activation. In addition, studies of triggering receptor expressed on myeloid cells 2-deficiency in AD mice revealed heterogeneous microglial reactions at different disease stages, complicating the therapeutic strategy for AD. In this paper, we describe the relationship between genetic and environmental risk factors and the roles of microglia in AD pathogenesis, based on studies performed in human patients and animal models. We also discuss the mechanisms of inflammasomes and neurotransmitters in microglia, which accelerate the development of amyloid-β and tau pathology.

RUNX1 and the endothelial origin of blood

The transcription factor RUNX1 is required in the embryo for formation of the adult hematopoietic system. Here, we describe the seminal findings that led to the discovery of RUNX1 and of its critical role in blood cell formation in the embryo from hemogenic endothelium (HE). We also present RNA-sequencing data demonstrating that HE cells in different anatomic sites, which produce hematopoietic progenitors with dissimilar differentiation potentials, are molecularly distinct. Hemogenic and non-HE cells in the yolk sac are more closely related to each other than either is to hemogenic or non-HE cells in the major arteries. Therefore, a major driver of the different lineage potentials of the committed erythro-myeloid progenitors that emerge in the yolk sac versus hematopoietic stem cells that originate in the major arteries is likely to be the distinct molecular properties of the HE cells from which they are derived. We used bioinformatics analyses to predict signaling pathways active in arterial HE, which include the functionally validated pathways Notch, Wnt, and Hedgehog. We also used a novel bioinformatics approach to assemble transcriptional regulatory networks and predict transcription factors that may be specifically involved in hematopoietic cell formation from arterial HE, which is the origin of the adult hematopoietic system.

Loss of Aquaporin-3 in Placenta and Fetal Membranes Induces Growth Restriction in Mice.

Aquaporin (AQP) 3, a facilitated transporter of water and glycerol, expresses in placenta and fetal membranes, but the detailed localization and function of AQP3 in placenta remain unclear. To elucidate a role of AQP3 in placenta, we defined the expression and cellular localization of AQP3 in placenta and fetal membranes, and investigated the structural and functional differences between wild-type and AQP3 null mice. Gestational sacs were removed during mid-gestational period and amniotic fluid was aspirated for measurements of volume and composition. Fetuses with attached placenta and fetal membranes were weighed and processed for histological assessment. AQP3 strongly expressed in basolateral membrane of visceral yolk sac cells of fetal membrane, the syncytiotrophoblasts of the labyrinthine placenta and fetal nucleated red blood cell membrane. Mice lacking AQP3 did not exhibit a significant defect in differentiation of trophoblast stem cells and normal placentation. However, AQP3 null fetuses were smaller than their control litter mates in spite of a decrease in litter size. The total amniotic fluid volume per gestational sac was reduced, but the amniotic fluid-to-fetal weight ratio was increased in AQP3 null mice compared with wild-type mice. Glycerol, free fatty acid and triglyceride levels in amniotic fluid of AQP3 null mice were significantly reduced, whereas lactate level increased when compared to those of wild-type mice. These results suggest a role for AQP3 in supplying nutrients from yolk sac and maternal blood to developing fetus by facilitating transport of glycerol in addition to water, and its implication for the fetal growth in utero.

Canine Placenta Recellularized Using Yolk Sac Cells with Vascular Endothelial Growth Factor.

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

Deficient Vitamin E Uptake During Development Impairs Neural Tube Closure in Mice Lacking Lipoprotein Receptor SR-BI

SR-BI is the main receptor for high density lipoproteins (HDL) and mediates the bidirectional transport of lipids, such as cholesterol and vitamin E, between these particles and cells. During early development, SR-BI is expressed in extraembryonic tissue, specifically in trophoblast giant cells in the parietal yolk sac. We previously showed that approximately 50% of SR-BI−/− embryos fail to close the anterior neural tube and develop exencephaly, a perinatal lethal condition. Here, we evaluated the role of SR-BI in embryonic vitamin E uptake during murine neural tube closure. Our results showed that SR-BI−/− embryos had a very low vitamin E content in comparison to SR-BI+/+ embryos. Whereas SR-BI−/− embryos with closed neural tubes (nSR-BI−/−) had high levels of reactive oxygen species (ROS), intermediate ROS levels between SR-BI+/+ and nSR-BI−/− embryos were detected in SR-BI−/− with NTD (NTD SR-BI−/−). Reduced expression of Pax3, Alx1 and Alx3 genes was found in NTD SR-BI−/− embryos. Maternal α-tocopherol dietary supplementation prevented NTD almost completely (from 54% to 2%, p < 0.001) in SR-BI−/− embryos and normalized ROS and gene expression levels. In sum, our results suggest the involvement of SR-BI in the maternal provision of embryonic vitamin E to the mouse embryo during neural tube closure.

Cellular uptake of proMMP-2:TIMP-2 complexes by the endocytic receptor megalin/LRP-2

Matrix metalloproteinases (MMPs) are regulated at multiple transcriptional and post-transcriptional levels, among which receptor-mediated endocytic clearance. We previously showed that low-density lipoprotein receptor-related protein-1 (LRP-1) mediates the clearance of a complex between the zymogen form of MMP-2 (proMMP-2) and tissue inhibitor of metalloproteinases, TIMP-2, in HT1080 human fibrosarcoma cells. Here we show that, in BN16 rat yolk sac cells, proMMP-2:TIMP-2 complex is endocytosed through a distinct LRP member, megalin/LRP-2. Addition of receptor-associated protein (RAP), a natural LRP antagonist, caused accumulation of endogenous proMMP-2 and TIMP-2 in conditioned media. Incubation with RAP also inhibited membrane binding and cellular uptake of exogenous iodinated proMMP-2:TIMP-2. Moreover, antibodies against megalin/LRP-2, but not against LRP-1, inhibited binding of proMMP-2:TIMP-2 to BN16 cell surface. BIAcore analysis confirmed direct interaction between the complex and megalin/LRP-2. Conditional renal invalidation of megalin/LRP-2 in mice resulted in accumulation of proMMP-2 and TIMP-2 in their urine, highlighting the physiological relevance of the binding. We conclude that megalin/LRP-2 can efficiently mediate cell-surface binding and endocytosis of proMMP-2:TIMP-2 complex. Therefore megalin/LRP-2 can be considered as a new actor in regulation of MMP-2 activity, an enzyme crucially involved in many pathological processes.

Fetal Membranes-Derived Stem Cells Microenvironment.

Recently, the regenerative medicine has been trying to congregate different areas such as tissue engineering and cellular therapy, in order to offer effective treatments to overcome several human and veterinary medical problems. In this regard, fetal membranes have been proposed as a powerful source for obtainment of multipotent stem cells with low immunogenicity, anti-inflammatory properties and nontumorigenicity properties for the treatment of several diseases, including replacing cells lost due to tissue injuries or degenerative diseases. Morpho-physiological data have shown that fetal membranes, especially the yolk sac and amnion play different functions according to the gestational period, which are direct related to the features of the microenvironment that their cells are subject. The characteristics of the microenvironment affect or controls important cellular events involved with proliferation, division and maintenance of the undifferentiated stage or differentiation, especially acting on the extracellular matrix components. Considering the importance of the microenvironment and the diversity of embryonic and fetal membrane-derived stem cells, this chapter will addressed advances in the isolation, phenotyping, characteristics of the microenvironment, and applications of yolk sac and amniotic membrane-derived stem cells for human and veterinary regenerative medicine.

Hemangioblast, hemogenic endothelium, and primitive versus definitive hematopoiesis.

The types of progenitors generated during the successive stages of embryonic blood development are now fairly well characterized. The terminology used to describe these waves, however,can still be confusing. What is truly primitive? What is uniquely definitive? These questions become even more challenging to answer when blood progenitors are derived invitro upon the differentiation of embryonic stem cells or induced pluripotent stem cells. Similarly, the cellular origin of these blood progenitors can be controversial. Are all blood cells, including the primitive wave, derived from hemogenic endothelium? Is the hemangioblast an invitro artifact or is this mesoderm entity also present in the developing embryo? Here, we discuss the latest findings and propose some consensus relating to these controversial issues.

Identifying the Molecular Mechanisms By Which Disruptor of Telomere Silencing 1-like (DOT1L), a Histone 3, Lysine 79 (H3K79) Methyltransferase, Regulates Mammalian Hematopoiesis

Disruptor of Telomere silencing 1-Like (DOT1L), is a histone 3, lysine 79 (H3K79) methyltransferase that has been implicated in multiple processes, including activation of transcription, regulation of the cell cycle, leukemogenesis, and mouse embryonic development. In previous studies, we found that Dot1L deficiency results in an erythropoietic defect, leading to lethal anemia at around mid-gestation (Feng et al., 2010, Blood). The precise molecular mechanism(s) by which DOT1L regulates embryonic hematopoiesis has not yet been elucidated and is the overall objective of this study. DOT1L is a large protein (1540aa), and it is involved in several, diverse processes. However, its only documented activity has been as an intrinsic, histone methyltransferase. Additional functional domains of the protein might be responsible for its diverse activities, including murine hematopoiesis. We sought to determine whether the methyltransferase activity of DOT1L is essential for hematopoiesis.To test this hypothesis, we developed a Dot1L methyltransferase mutant (Dot1L MM) mouse line. Using the Cas9/CRISPR system, we created a Dot1L point mutation in cultured murine embryonic stem cells (mESC). These mESCs contained a wildtype allele, and the second had a single amino acid change in the methyltransferase domain of Dot1L, thereby eliminating its methyltransferase activity, but preserving the rest of the protein. We injected these mutant mESCs into blastocysts to produce chimeric mice. The chimeras containing the methyltransferase mutation were back crossed onto the C57/BL6 background, producing male and female offspring heterozygous for the mutation. Through intercrosses of the F1 generation, we found that the Dot1L MM mice displayed an embryonic lethality between embryonic days 10.5 and 13.5, similar to the Dot1L knockout mice, as reported in our previous studies.We additionally performed ex vivo blood differentiation assays and extensively self-renewing erythroblast (ESRE) cultures using E10.5 yolk sacs from Dot1L MM and knockout mice. Our data showed that the Dot1L MM and knockout yolk sacs display similar phenotypes. In blood differentiation cultures, Dot1L knockout and MM yolk sac cells form the same types of hematopoietic colonies as in wildtype (both erythroid and myeloid), but there is a decrease in colony size and number compared to the wildtype. In the ESRE cultures, Dot1L knockout and MM yolk sac cells form significantly fewer ESREs and have increased cell death compared to wildtype. Strikingly, the cells in these cultures also exhibit a profound genomic instability, implicating DOT1L methyltransferase activity in maintenance of the genome as well as the viability of hematopoietic progenitors. These results suggest that the methyltransferase activity of DOT1L plays a predominant role in the activity of the protein as a whole, and is responsible for its function in facilitating early murine hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Identification of novel genetic markers for mouse yolk sac cells by using microarray analyses

The mouse embryonic yolk sac consists of a visceral yolk sac (VYS) and parietal yolk sac (PYS), and may function as a materno-fetal exchange system for nutrients and wastes, and physical protector for the embryo/fetus. The present study was undertaken to characterize gene expression of the VYS and PYS endodermal cells, and to identify their novel genetic markers from microarray data. Apoa4, Lrp2, Fxyd2, Slc34a3 and Entpd2 were predominantly expressed in VYS epithelial cells. Gkn2 and Pga5 were selected as markers for PYS cells. These genetic markers may be useful for characterization of murine yolk sacs during development.

The expression of embryonic globin mRNA in a severely anemic mouse model induced by treatment with nitrogen-containing bisphosphonate.

BackgroundMammalian erythropoiesis can be divided into two distinct types, primitive and definitive, in which new cells are derived from the yolk sac and hematopoietic stem cells, respectively. Primitive erythropoiesis occurs within a restricted period during embryogenesis. Primitive erythrocytes remain nucleated, and their hemoglobins are different from those in definitive erythrocytes. Embryonic type hemoglobin is expressed in adult animals under genetically abnormal condition, but its later expression has not been reported in genetically normal adult animals, even under anemic conditions. We previously reported that injecting animals with nitrogen-containing bisphosphonate (NBP) decreased erythropoiesis in bone marrow (BM). Here, we induced severe anemia in a mouse model by injecting NBP injection in combination with phenylhydrazine (PHZ), and then we analyzed erythropoiesis and the levels of different types of hemoglobin.MethodsSplenectomized mice were treated with NBP to inhibit erythropoiesis in BM, and with PHZ to induce hemolytic anemia. We analyzed hematopoietic sites and peripheral blood using morphological and molecular biological methods.ResultsCombined treatment of splenectomized mice with NBP and PHZ induced critical anemia compared to treatment with PHZ alone, and numerous nucleated erythrocytes appeared in the peripheral blood. In the BM, immature CD71-positive erythroblasts were increased, and extramedullary erythropoiesis occurred in the liver. Furthermore, embryonic type globin mRNA was detected in both the BM and the liver. In peripheral blood, spots that did not correspond to control hemoglobin were observed in 2D electrophoresis. ChIP analyses showed that KLF1 and KLF2 bind to the promoter regions of β-like globin. Wine-colored capsuled structures were unexpectedly observed in the abdominal cavity, and active erythropoiesis was also observed in these structures.ConclusionThese results indicate that primitive erythropoiesis occurs in adult mice to rescue critical anemia because primitive erythropoiesis does not require macrophages as stroma whereas macrophages play a pivotal role in definitive erythropoiesis even outside the medulla. The cells expressing embryonic hemoglobin in this study were similar to primitive erythrocytes, indicating the possibility that yolk sac-derived primitive erythroid cells may persist into adulthood in mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12878-016-0041-0) contains supplementary material, which is available to authorized users.

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: HNF1β, 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 HNF1β-, 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.

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.

Diabetes affects rat visceral yolk sac cells viability and cell markers expression

Rodents have a well-developed visceral yolk sac (VYS) that acts as an active region for metabolic exchange and nutrition uptake until final fetal development in rats and may be affected by diabetes . Using Wistar rats, diabetes was induced by a single injection of alloxan at gestational day 8 (8 gd) and at 15 gd the collection of VYS was made. Flow cytometry was performed for VYS cell characterization, determination of mitochondrial activity, cell proliferation, DNA ploidy, cell cycle phases and caspase-3 activity. Fetal weight was reduced in the diabetic group. CD34, CCR2, and OCT3/4 expressions were significantly reduced, and CD90, CD117, and CD14 expressions were increased in the diabetic group. VYS cells with inactive mitochondria, activated caspase-3, and low proliferation were present in the diabetic condition. Severe hyperglycemia due to maternal diabetes was shown to have negative effects on pregnancy, VYS viability, and cell marker expression.

Neuropilin-2 genomic elements drive cre recombinase expression in primitive blood, vascular and neuronal lineages.

We have established a novel Cre mouse line, using genomic elements encompassing the Nrp2 locus, present within a bacterial artificial chromosome clone. By crossing this Cre driver line to R26R LacZ reporter mice, we have documented the temporal expression and lineage traced tissues in which Cre is expressed. Nrp2-Cre drives expression in primitive blood cells arising from the yolk sac, venous and lymphatic endothelial cells, peripheral sensory ganglia, and the lung bud. This mouse line will provide a new tool to researchers wishing to study the development of various tissues and organs in which this Cre driver is expressed, as well as allow tissue-specific knockout of genes of interest to study protein function. This work also presents the first evidence for expression of Nrp2 protein in a mesodermal progenitor with restricted hematopoietic potential, which will significantly advance the study of primitive erythropoiesis. genesis 53:709-717, 2015. © 2015 Wiley Periodicals, Inc.

Elevation of urinary methylmolonic acid induces the suppression of megalin-mediated endocytotic cycles during vitamin B12 deficiency

Megalin is a scavenger receptor that serves in the endocytosis of a highly diverse group of ligands that includes Vitamin B12. We found an accumulation of megalin closed to apical region in renal proximal tubule cells of Vitamin B12-deficient rats. Interestingly, Vitamin B12 levels also controlled resorption of renal retinol binding protein. Using L2 yolk sac cells, megalin localized to the submembrane compartment by methylmalonic acid (MMA), which accumulates during vitamin B12 deficiency. In addition, MMA inhibited megalin-mediated endocytosis via YWTD repeats motif in an ectodomain of megalin. Therefore, megalin endocytosis may be regulated by MMA.