Endothelin receptor-A mediates degradation of the glomerular endothelial surface layer via pathologic crosstalk between activated podocytes and glomerular endothelial cells.

Emerging evidence of crosstalk between glomerular cells in pathological settings provides opportunities for novel therapeutic discovery. Here we investigated underlying mechanisms of early events leading to filtration barrier defects of podocyte and glomerular endothelial cell crosstalk in the mouse models of primary podocytopathy (podocyte specific transforming growth factor-ß receptor 1 signaling activation) or Adriamycin nephropathy. We found that glomerular endothelial surface layer degradation and albuminuria preceded podocyte foot process effacement. These abnormalities were prevented by endothelin receptor-A antagonism and mitochondrial reactive oxygen species scavenging. Additional studies confirmed increased heparanase and hyaluronoglucosaminidase gene expression in glomerular endothelial cells in response to podocyte-released factors and to endothelin-1. Atomic force microscopy measurements showed a significant reduction in the endothelial surface layer by endothelin-1 and podocyte-released factors, which could be prevented by endothelin receptor-A but not endothelin receptor-B antagonism. Thus, our studies provide evidence of early crosstalk between activated podocytes and glomerular endothelial cells resulting in loss of endothelial surface layer, glomerular endothelial cell injury and albuminuria. Hence, activation of endothelin-1-endothelin receptor-A and mitochondrial reactive oxygen species contribute to the pathogenesis of primary podocytopathies in experimental focal segmental glomerulosclerosis.

Gene expression profiles of glomerular endothelial cells support their role in the glomerulopathy of diabetic mice.

Endothelial dysfunction promotes the pathogenesis of diabetic nephropathy (DN), which is considered to be an early event in disease progression. However, the molecular changes associated with glomerular endothelial cell (GEC) injury in early DN are not well defined. Most gene expression studies have relied on the indirect assessment of GEC injury from isolated glomeruli or renal cortices. Here, we present transcriptomic analysis of isolated GECs, using streptozotocin-induced diabetic wildtype (STZ-WT) and diabetic eNOS-null (STZ-eNOS-/-) mice as models of mild and advanced DN, respectively. GECs of both models in comparison to their respective nondiabetic controls showed significant alterations in the regulation of apoptosis, oxidative stress, and proliferation. The extent of these changes was greater in STZ-eNOS-/- than in STZ-WT GECs. Additionally, genes in STZ-eNOS-/- GECs indicated further dysregulation in angiogenesis and epigenetic regulation. Moreover, a biphasic change in the number of GECs, characterized by an initial increase and subsequent decrease over time, was observed only in STZ-eNOS-/- mice. This is consistent with an early compensatory angiogenic process followed by increased apoptosis, leading to an overall decrease in GEC survival in DN progression. From the genes altered in angiogenesis in STZ-eNOS-/- GECs, we identified potential candidate genes, Lrg1 and Gpr56, whose function may augment diabetes-induced angiogenesis. Thus, our results support a role for GEC in DN by providing direct evidence for alterations of GEC gene expression and molecular pathways. Candidate genes of specific pathways, such as Lrg1 and Gpr56, can be further explored for potential therapeutic targeting to mitigate the initiation and progression of DN.

Expression of podocalyxin separates the hematopoietic and vascular potentials of mouse embryonic stem cell-derived mesoderm.

In the mouse embryo and differentiating embryonic stem cells, the hematopoietic, endothelial, and cardiomyocyte lineages are derived from Flk1+ mesodermal progenitors. Here, we report that surface expression of Podocalyxin (Podxl), a member of the CD34 family of sialomucins, can be used to subdivide the Flk1+ cells in differentiating embryoid bodies at day 4.75 into populations that develop into distinct mesodermal lineages. Definitive hematopoietic potential was restricted to the Flk1+Podxl+ population, while the Flk1-negative Podxl+ population displayed only primitive erythroid potential. The Flk1+Podxl-negative population contained endothelial cells and cardiomyocyte potential. Podxl expression distinguishes Flk1+ mesoderm populations in mouse embryos at days 7.5, 8.5, and 9.5 and is a marker of progenitor stage primitive erythroblasts. These findings identify Podxl as a useful tool for separating distinct mesodermal lineages.

The heme exporter Flvcr1 regulates expansion and differentiation of committed erythroid progenitors by controlling intracellular heme accumulation.

Feline leukemia virus subgroup C receptor 1 (Flvcr1) encodes two heme exporters: FLVCR1a, which localizes to the plasma membrane, and FLVCR1b, which localizes to mitochondria. Here, we investigated the role of the two Flvcr1 isoforms during erythropoiesis. We showed that, in mice and zebrafish, Flvcr1a is required for the expansion of committed erythroid progenitors but cannot drive their terminal differentiation, while Flvcr1b contributes to the expansion phase and is required for differentiation. FLVCR1a-down-regulated K562 cells have defective proliferation, enhanced differentiation, and heme loading in the cytosol, while FLVCR1a/1b-deficient K562 cells show impairment in both proliferation and differentiation, and accumulate heme in mitochondria. These data support a model in which the coordinated expression of Flvcr1a and Flvcr1b contributes to control the size of the cytosolic heme pool required to sustain metabolic activity during the expansion of erythroid progenitors and to allow hemoglobinization during their terminal maturation. Consistently, reduction or increase of the cytosolic heme rescued the erythroid defects in zebrafish deficient in Flvcr1a or Flvcr1b, respectively. Thus, heme export represents a tightly regulated process that controls erythropoiesis.

A conditional mutant allele for analysis of Mixl1 function in the mouse.

Mixl1 is the only member of the Mix/Bix homeobox gene family identified in mammals. During mouse embryogenesis, Mixl1 is first expressed at embryonic day (E)5.5 in cells of the visceral endoderm (VE). At the time of gastrulation, Mixl1 expression is detected in the vicinity of the primitive streak. Mixl1 is expressed in cells located within the primitive streak, in nascent mesoderm cells exiting the primitive streak, and in posterior VE overlying the primitive streak. Genetic ablation of Mixl1 in mice has revealed its crucial role in mesoderm and endoderm cell specification and tissue morphogenesis during early embryonic development. However, the early lethality of the constitutive Mixl1(-/-) mutant precludes the study of its role at later stages of embryogenesis and in adult mice. To circumvent this limitation, we have generated a conditional Mixl1 allele (Mixl1(cKO) that permits temporal as well as spatial control of gene ablation. Animals homozygous for the Mixl1(cKO) conditional allele were viable and fertile. Mixl1(KO/KO) embryos generated by crossing of Mixl1(cKO/cKO) mice with Sox2-Cre or EIIa-Cre transgenic mice were embryonic lethal at early somite stages. By contrast to wild-type embryos, Mixl1(KO/KO) embryos contained no detectable Mixl1, validating the Mixl1(cKO) as a protein null after Cre-mediated excision. Mixl1(KO/KO) embryos resembled the previously reported Mixl1(-/-) mutant phenotype. Therefore, the Mixl1 cKO allele provides a tool for investigating the temporal and tissue-specific requirements for Mixl1 in the mouse.

The embryonic origins of erythropoiesis in mammals.

Erythroid (red blood) cells are the first cell type to be specified in the postimplantation mammalian embryo and serve highly specialized, essential functions throughout gestation and postnatal life. The existence of 2 developmentally and morphologically distinct erythroid lineages, primitive (embryonic) and definitive (adult), was described for the mammalian embryo more than a century ago. Cells of the primitive erythroid lineage support the transition from rapidly growing embryo to fetus, whereas definitive erythrocytes function during the transition from fetal life to birth and continue to be crucial for a variety of normal physiologic processes. Over the past few years, it has become apparent that the ontogeny and maturation of these lineages are more complex than previously appreciated. In this review, we highlight some common and distinguishing features of the red blood cell lineages and summarize advances in our understanding of how these cells develop and differentiate throughout mammalian ontogeny.

Concise Review: early embryonic erythropoiesis: not so primitive after all.

In the developing embryo, hematopoiesis begins with the formation of primitive erythroid cells (EryP), a distinct and transient red blood cell lineage. EryP play a vital role in oxygen delivery and in generating shear forces necessary for normal vascular development. Progenitors for EryP arise as a cohort within the blood islands of the mammalian yolk sac at the end of gastrulation. As a strong heartbeat is established, nucleated erythroblasts begin to circulate and to mature in a stepwise, nearly synchronous manner. Until relatively recently, these cells were thought to be "primitive" in that they seemed to more closely resemble the nucleated erythroid cells of lower vertebrates than the enucleated erythrocytes of mammals. It is now known that mammalian EryP do enucleate, but not until several days after entering the bloodstream. I will summarize the common and distinguishing characteristics of primitive versus definitive (adult-type) erythroid cells, review the development of EryP from the emergence of their progenitors through maturation and enucleation, and discuss pluripotent stem cells as models for erythropoiesis. Erythroid differentiation of both mouse and human pluripotent stem cells in vitro has thus far reproduced early but not late red blood cell ontogeny. Therefore, a deeper understanding of cellular and molecular mechanisms underlying the differences and similarities between the embryonic and adult erythroid lineages will be critical to improving methods for production of red blood cells for use in the clinic.

FOXO3-mTOR metabolic cooperation in the regulation of erythroid cell maturation and homeostasis.

Ineffective erythropoiesis is observed in many erythroid disorders including ß-thalassemia and anemia of chronic disease in which increased production of erythroblasts that fail to mature exacerbate the underlying anemias. As loss of the transcription factor FOXO3 results in erythroblast abnormalities similar to the ones observed in ineffective erythropoiesis, we investigated the underlying mechanisms of the defective Foxo3(-/-) erythroblast cell cycle and maturation. Here we show that loss of Foxo3 results in overactivation of the JAK2/AKT/mTOR signaling pathway in primary bone marrow erythroblasts partly mediated by redox modulation. We further show that hyperactivation of mTOR signaling interferes with cell cycle progression in Foxo3 mutant erythroblasts. Importantly, inhibition of mTOR signaling, in vivo or in vitro enhances significantly Foxo3 mutant erythroid cell maturation. Similarly, in vivo inhibition of mTOR remarkably improves erythroid cell maturation and anemia in a model of ß-thalassemia. Finally we show that FOXO3 and mTOR are likely part of a larger metabolic network in erythroblasts as together they control the expression of an array of metabolic genes some of which are implicated in erythroid disorders. These combined findings indicate that a metabolism-mediated regulatory network centered by FOXO3 and mTOR control the balanced production and maturation of erythroid cells. They also highlight physiological interactions between these proteins in regulating erythroblast energy. Our results indicate that alteration in the function of this network might be implicated in the pathogenesis of ineffective erythropoiesis.

Analysis of primitive erythroid cell proliferation and enucleation using a cyan fluorescent reporter in transgenic mice.

Primitive erythropoiesis is a vital process for mammalian embryonic development. Here we report the generation and characterization of a new transgenic mouse line that expresses a histone H2B-CFP fusion protein in the nuclei of primitive erythroid cells. We demonstrate the potential of this ε-globin-histone H2B-CFP line for multicolor imaging and flow cytometry analysis. The ε-globin-H2B-CFP line was used to analyze the cell cycle distribution and proliferation of CFP-expressing primitive erythroblasts from E8.5-E13.5. We also evaluated phagocytosis of extruded CFP-positive nuclei by macrophages in fetal liver and placenta. The ε-globin-H2B-CFP transgenic mouse line adds to the available tools for studying the development of the primitive erythroid lineage.

BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity.

The visceral endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the primitive endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic ENdoderm (XEN) cells can be propagated in vitro, XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signaling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation and therefore represent a valuable tool for investigating PrE lineage differentiation.

Erythroid development in the mammalian embryo.

Erythropoiesis is the process by which progenitors for red blood cells are produced and terminally differentiate. In all vertebrates, two morphologically distinct erythroid lineages (primitive, embryonic, and definitive, fetal/adult) form successively within the yolk sac, fetal liver, and marrow and are essential for normal development. Red blood cells have evolved highly specialized functions in oxygen transport, defense against oxidation, and vascular remodeling. Here we review key features of the ontogeny of red blood cell development in mammals, highlight similarities and differences revealed by genetic and gene expression profiling studies, and discuss methods for identifying erythroid cells at different stages of development and differentiation.

Single-lineage transcriptome analysis reveals key regulatory pathways in primitive erythroid progenitors in the mouse embryo.

Primitive erythroid (EryP) progenitors are the first cell type specified from the mesoderm late in gastrulation. We used a transgenic reporter to image and purify the earliest blood progenitors and their descendants from developing mouse embryos. EryP progenitors exhibited remarkable proliferative capacity in the yolk sac immediately before the onset of circulation, when these cells comprise nearly half of all cells of the embryo. Global expression profiles generated at 24-hour intervals from embryonic day 7.5 through 2.5 revealed 2 abrupt changes in transcript diversity that coincided with the entry of EryPs into the circulation and with their late maturation and enucleation, respectively. These changes were paralleled by the expression of critical regulatory factors. Experiments designed to test predictions from these data demonstrated that the Wnt-signaling pathway is active in EryP progenitors, which display an aerobic glycolytic profile and the numbers of which are regulated by transforming growth factor-ß1 and hypoxia. This is the first transcriptome assembled for a single hematopoietic lineage of the embryo over the course of its differentiation.

Dose-dependent regulation of primitive erythroid maturation and identity by the transcription factor Eklf.

The primitive erythroid (EryP) lineage is the first to differentiate during mammalian embryogenesis. Eklf/Klf1 is a transcriptional regulator that is essential for definitive erythropoiesis in the fetal liver. Dissection of the role(s) of Eklf within the EryP compartment has been confounded by the simultaneous presence of EryP and fetal liver-derived definitive erythroid (EryD) cells in the blood. To address this problem, we have distinguished EryP from their definitive counterparts by crossing Eklf(+/-) mutant and ε-globin::histone H2B-GFP transgenic mice. Eklf-deficient EryP exhibit membrane ruffling and a failure to acquire the typical discoidal erythroid shape but they can enucleate. Flow cytometric analyses of H2B-GFP(+) EryP revealed that Eklf heterozygosity results in the loss of Ter119 surface expression on EryP but not on EryD. Null mutation of Eklf resulted in abnormal expression of a range of surface proteins by EryP. In particular, several megakaryocyte markers were ectopically expressed by maturing Eklf-null EryP. Unexpectedly, the platelet tetraspanin CD9 was detected on nucleated wild-type EryP but not on mature EryD and thus provides a useful marker for purifying circulating EryP. We conclude that Eklf gene dosage is crucial for regulating the surface phenotype and molecular identity of maturing primitive erythroid cells.

The fetal liver is a niche for maturation of primitive erythroid cells.

Primitive erythroid cells (EryP) are the earliest differentiated cell type of the mammalian embryo. They appear in the yolk sac by embryonic day 7.5, begin to enter the embryonic circulation 2 days later and continue to mature in a stepwise and synchronous fashion. Like their adult counterparts, EryP enucleate. However, EryP circulate throughout the embryo for several days before the first enucleated forms can be identified in the blood. We have used transgenic mouse lines in which GFP marks EryP to investigate this seemingly long lag and have identified a previously unrecognized developmental niche for EryP maturation. After exiting the yolk sac, EryP begin to express cell adhesion proteins, including alpha4, alpha5, and beta1 integrins, on their surface and migrate into the fetal liver (FL), where they interact with macrophages within erythroblastic islands. Binding of EryP to FL macrophages in vitro is stage-specific and partly depends on VCAM-1. The ability to tag and track EryP nuclei using a transgenic mouse line expressing an H2B-EGFP fusion allowed us to identify and characterize extruded EryP nuclei and to demonstrate that molecules such as alpha4, alpha5, and beta1 integrins are redistributed onto the plasma membrane surrounding the extruding nucleus. FL macrophages engulf extruded EryP nuclei in cocultures and in the native FL in vivo. We conclude that EryP home to, complete their maturation, and enucleate within the FL, a tissue that is just developing as EryP begin to circulate. Our observations suggest a simple solution for a puzzling aspect of the development of the primitive erythroid lineage.

Generation of Transgenic Fluorescent Reporter Lines for Studying Hematopoietic Development in the Mouse.

Hematopoiesis in the mouse and other mammals occurs in several waves and arises from distinct anatomic sites. Transgenic mice expressing fluorescent reporter proteins at various points in the hematopoietic hierarchy, from hematopoietic stem cell to more restricted progenitors to each of the final differentiated cell types, have provided valuable tools for tagging, tracking, and isolating these cells. In this chapter, we discuss general considerations in designing a transgene, survey available fluorescent probes, and describe methods for confirming and analyzing transgene expression in the hematopoietic tissues of the embryo, fetus, and postnatal/adult animal.

Developmental niches for embryonic erythroid cells.

Primitive erythroid cells (EryP) are the first differentiated cell type to be specified during mammalian embryogenesis. EryP arise from a pool of lineage-restricted progenitors in the yolk sac (YS) and then enter the newly formed embryonic circulation to mature in a stepwise, synchronous fashion. Numbering in the millions in the mid-gestation mouse embryo, EryP are the dominant circulating blood cell prior to the rapid generation of adult-type definitive erythroid (EryD) cells in the fetal liver. The identification of maturational events in this lineage presented a significant challenge, as EryD begin to outnumber EryP in the bloodstream from approximately E14.5 onwards. We used human epsilon-globin gene regulatory elements to drive lineage-specific expression of a histone-H2B::EGFP fusion protein, allowing us to label the chromatin of EryP during their development and to track and quantify EryP nuclei following their expulsion from the cell. Using this transgenic fluorescent reporter mouse line, we have monitored primitive erythropoiesis in three distinct niches: the YS, where EryP progenitors arise; the circulation, where EryP continue to divide and mature; and the fetal liver, where EryP complete the terminal stages of their differentiation.

Transcriptional activation by the Mixl1 homeodomain protein in differentiating mouse embryonic stem cells.

Members of the Mix/Bix family of paired class homeobox genes play important roles in the development of vertebrate mesoderm and endoderm. The single Mix/Bix family member identified in the mouse, Mix-like 1 (Mixl1), is required for mesendoderm patterning during gastrulation and promotes mesoderm formation and hematopoiesis in embryonic stem cell (ESC)-derived embryoid bodies. Despite its crucial functions the transcriptional activity and targets of Mixl1 have not been well described. To investigate the molecular mechanisms of Mixl1-mediated transcriptional regulation, we have characterized the DNA-binding specificity and transcriptional properties of this homeodomain protein in differentiating ESCs. Mixl1 binds preferentially as a dimer to an 11-base pair (bp) Mixl1 binding sequence (MBS) that contains two inverted repeats separated by a 3-bp spacer. The MBS mediates transcriptional activation by Mixl1 in both NIH 3T3 cells and in a new application of an inducible ESC differentiation system. Consistent with our previous observation that early induction of Mixl1 expression in ESCs results in premature activation of Goosecoid (Gsc), we have found that Mixl1 occupies two variant MBSs within and activates transcription from the Gsc promoter in vitro and in vivo. These results strongly suggest that Gsc is a direct target gene of Mixl1 during embryogenesis. STEM CELLS 2009;27:2884-2895.

Embryonic stromal clones reveal developmental regulators of definitive hematopoietic stem cells.

Hematopoietic stem cell (HSC) self-renewal and differentiation is regulated by cellular and molecular interactions with the surrounding microenvironment. During ontogeny, the aorta-gonad-mesonephros (AGM) region autonomously generates the first HSCs and serves as the first HSC-supportive microenvironment. Because the molecular identity of the AGM microenvironment is as yet unclear, we examined two closely related AGM stromal clones that differentially support HSCs. Expression analyses identified three putative HSC regulatory factors, beta-NGF (a neurotrophic factor), MIP-1gamma (a C-C chemokine family member) and Bmp4 (a TGF-beta family member). We show here that these three factors, when added to AGM explant cultures, enhance the in vivo repopulating ability of AGM HSCs. The effects of Bmp4 on AGM HSCs were further studied because this factor acts at the mesodermal and primitive erythropoietic stages in the mouse embryo. In this report, we show that enriched E11 AGM HSCs express Bmp receptors and can be inhibited in their activity by gremlin, a Bmp antagonist. Moreover, our results reveal a focal point of Bmp4 expression in the mesenchyme underlying HSC containing aortic clusters at E11. We suggest that Bmp4 plays a relatively late role in the regulation of HSCs as they emerge in the midgestation AGM.

Embryonic fates for extraembryonic lineages: New perspectives.

The prevailing view of the functions of the extraembryonic lineages of the mammalian embryo has been that they serve solely to support its intrauterine development. In recent years, a number of studies have suggested that the extraembryonic mesoderm and visceral endoderm in fact contribute cells to tissues of the developing animal. In this mini-review, we discuss evidence that the yolk sac is an early source of hematopoietic stem and progenitor cells and that the cells of the visceral endoderm, once thought to be segregated solely to the yolk sac, constitute a subpopulation of cells within the developing gut tube and perhaps other endodermal structures. Fascinating questions remain to be addressed and are likely to establish a new paradigm for studying early mammalian development. Understanding the processes that give rise to stem cell populations in development may lead to advances in stem cell therapies and regenerative medicine.