Yolk Sac Vasculature Research Articles

HoxA3 is an apical regulator of haemogenic endothelium

During development, haemogenesis occurs invariably at sites of vasculogenesis. Between embryonic day (E) 9.5 and E10.5 in mice, endothelial cells in the caudal part of the dorsal aorta generate haematopoietic stem cells and are referred to as haemogenic endothelium. The mechanisms by which haematopoiesis is restricted to this domain, and how the morphological transformation from endothelial to haematopoietic is controlled are unknown. We show here that HoxA3, a gene uniquely expressed in the embryonic but not yolk sac vasculature, restrains haematopoietic differentiation of the earliest endothelial progenitors, and induces reversion of the earliest haematopoietic progenitors into CD41-negative endothelial cells. This reversible modulation of endothelial-haematopoietic state is accomplished by targeting key haematopoietic transcription factors for downregulation, including Runx1, Gata1, Gfi1B, Ikaros, and PU.1. Through loss-of-function, and gain-of-function epistasis experiments, and the identification of antipodally regulated targets, we show that among these factors, Runx1 is uniquely able to erase the endothelial program set up by HoxA3. These results suggest both why a frank endothelium does not precede haematopoiesis in the yolk sac, and why haematopoietic stem cell generation requires Runx1 expression only in endothelial cells.

Rapid remodeling of airway vascular architecture at birth

Recent advances have documented the development of lung vasculature before and after birth, but less is known of the growth and maturation of airway vasculature. We sought to determine whether airway vasculature changes during the perinatal period and when the typical adult pattern develops. On embryonic day 16.5 mouse tracheas had a primitive vascular plexus unlike the adult airway vasculature, but instead resembling the yolk sac vasculature. Soon after birth (P0), the primitive vascular plexus underwent abrupt and extensive remodeling. Blood vessels overlying tracheal cartilage rings regressed from P1 to P3 but regrew from P4 to P7 to form the hierarchical, segmented, ladder-like adult pattern. Hypoxia and HIF-1α were present in tracheal epithelium over vessels that survived but not where they regressed. These findings reveal the plasticity of airway vasculature after birth and show that these vessels can be used to elucidate factors that promote postnatal vascular remodeling and maturation.

Pulsatile shear and Gja5 modulate arterial identity and remodeling events during flow-driven arteriogenesis

In the developing chicken embryo yolk sac vasculature, the expression of arterial identity genes requires arterial hemodynamic conditions. We hypothesize that arterial flow must provide a unique signal that is relevant for supporting arterial identity gene expression and is absent in veins. We analyzed factors related to flow, pressure and oxygenation in the chicken embryo vitelline vasculature in vivo. The best discrimination between arteries and veins was obtained by calculating the maximal pulsatile increase in shear rate relative to the time-averaged shear rate in the same vessel: the relative pulse slope index (RPSI). RPSI was significantly higher in arteries than veins. Arterial endothelial cells exposed to pulsatile shear in vitro augmented arterial marker expression as compared with exposure to constant shear. The expression of Gja5 correlated with arterial flow patterns: the redistribution of arterial flow provoked by vitelline artery ligation resulted in flow-driven collateral arterial network formation and was associated with increased expression of Gja5. In situ hybridization in normal and ligation embryos confirmed that Gja5 expression is confined to arteries and regulated by flow. In mice, Gja5 (connexin 40) was also expressed in arteries. In the adult, increased flow drives arteriogenesis and the formation of collateral arterial networks in peripheral occlusive diseases. Genetic ablation of Gja5 function in mice resulted in reduced arteriogenesis in two occlusion models. We conclude that pulsatile shear patterns may be central for supporting arterial identity, and that arterial Gja5 expression plays a functional role in flow-driven arteriogenesis.

Modulation of Myocardin Function by the Ubiquitin E3 Ligase UBR5

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.

IgG is transported across the mouse yolk sac independently of FcγRIIb

While generally accepted that FcRn of the human syncytiotrophoblast and the mouse yolk sac endoderm is the major IgG transporter, the finding of a different Fc receptor FcgammaRIIb (RIIb) in the human placental endothelium has suggested the existence of an additional IgG transporter. Testing our hypothesis in mouse, we found that while RIIb is expressed in the yolk sac vasculature, IgG concentrations in fetuses of wild-type mice (RIIb(+/+)) and mice with a null mutation in the gene encoding RIIb (RIIb(-/-) mice) are not different, and we thus reject our hypothesis that yolk sac RIIb transports IgG in utero in the mouse. However, the capillary bed in the mouse yolk sac is structurally more complex than in human placenta, consisting of three types of cells: an RIIb-negative endothelium, a unique RIIb-bearing cell that also expresses 2 out of 4 macrophage markers but not endothelial cell or pericyte markers, and pericytes. As in the human placenta the b2 isoform of RIIb predominates in the mouse yolk sac. Remarkably only a single capillary channel rather than 2 channels with a loop is found in each yolk sac villus, which, along with intracapillary erythrocytes, suggests that blood flow is peristaltic, mediated by pericytes. It is not clear whether RIIb in the human placental villus might mediate an IgG transport function in light of the mouse yolk sac equivalent failing to do so.

Generation of mouse conditional and null alleles of the type III sodium-dependent phosphate cotransporter PiT-1.

Accelerated vascular calcification occurs in several human diseases including diabetes and chronic kidney disease (CKD). In patients with CKD, vascular calcification is highly correlated with elevated serum phosphate levels. In vitro, elevated concentrations of phosphate induced vascular smooth muscle cell matrix mineralization, and the inorganic phosphate transporter-1 (PiT-1), was shown to be required. To determine the in vivo role of PiT-1, mouse conditional and null alleles were generated. Here we show that the conditional allele, PiT-1(flox), which has loxP sites flanking exons 3 and 4, is homozygous viable. Cre-mediated recombination resulted in a null allele that is homozygous lethal. Examination of early embryonic development revealed that the PiT-1(Deltae3,4/Deltae3,4) embryos displayed anemia, a defect in yolk sac vasculature, and arrested growth. Thus, conditional and null PiT-1 mouse alleles have been successfully generated and PiT-1 has a necessary, nonredundant role in embryonic development.

Abstract 3665: Aggf1 Is Essential for Embryonic and Pathological Angiogenesis

Backgroud: AGGF1 is a newly identified angiogenic factor associated with vascular disease Klippel-Trenaunay syndrome. AGGF1 has been shown to promote angiogenesis as potently as VEGF in a chicken embryo angiogenesis assay; however, the physiological role of AGGF1 is unknown. Methods and Results: We explored the physiological function of AGGF1 in vivo by employing the gene-trapping strategy in mice. AGGF1−/− mice died by E8.5, suggesting that AGGF1 is essential for embryogenesis. AGGF1 heterozygosity resulted in 31% embryonic lethality. Further macroscopic analysis of embryos at different stages revealed growth retardation and defective vascular development in AGGF1+/− embryos. The major vascular phenotypes included failed development of large vessels and interconnected yolk sac vasculature. Interestingly, decreased phosphorylation of VEGFR2 (Tyr951) was detected in AGGF1+/− embryos compared to the wild type littermate controls, suggesting that AGGF1 may function through VEGFR2-mediated signaling during angiogenesis. Moreover, vascular pathologies of various internal organs were detected in older AGGF1+/− mice. Hemorrhages were frequently noted in brain, spleen and lung, and a chronic inflammatory change was readily detected in lungs in AGGF1+/− mice compared to the littermate controls. Permeability assay further revealed increased vascular permeability in old AGGF1+/− mice (50 – 60 weeks) compared to age- and sex-matched wild-type controls. A tumor angiogenesis model induced by two melanoma cell lines was employed to further assess the angiogenic function of AGGF1 in vivo. Tumor growth was significantly inhibited in AGGF1+/− mice. Histological examinations of the tumor sections revealed markedly reduced microvessel formation in tumors grown in AGGF1+/− mice. Moreover, vascular density quantified after PECAM1 immunostaining showed significant reduction in tumors grown in AGGF1+/− mice. Conclusions: AGGF1 is an important regulator for embryonic angiogenesis and vascular integrity. AGGF1 is also involved in tumor growth and pathological angiogenesis. These results provide for the first time the in vivo experimental evidence that AGGF1 is a potent angiogenic factor essential for both embryonic and pathological angiogenesis.

Human embryonic stem cell-derived hematoendothelial progenitors engraft chicken embryos

To investigate whether human embryonic stem cells (hESC) committed in culture into hematopoietic/endothelial cell progenitors can be further developed into mature blood and vascular cells following transplantation into chicken embryos. The yolk sac of 42- to 44-hour chicken embryos received yolk sac injections of unfractionated human embryoid body (hEB) cells, CD34-positive hEB cells, or CD34+CD45+ granulocyte colony-stimulating factor-mobilized human peripheral blood hematopoietic stem-progenitor cells. Human cells in the host were detected by flow cytometry and immunohistochemistry. All injected cell populations engrafted chicken hematopoietic organs, as assessed by detection of CD45+ cells in the spleen, bursa of Fabricius, and thymus. CD34+ day -10 hEB cells showed the highest efficiency for producing human CD45+ cells in the hosts and yielded human glycophorin A+ erythroid, CD13+ myeloid, and CD19+ lymphoid cells in the spleen and bursa of Fabricius. Spleen cells from chimeric embryos also contained human colony-forming units-granulocyte macrophage, as assessed in methylcellulose colony-forming assays. Human endothelial cells expressing vascular endothelial-cadherin, von Willebrand factor, CD31, and the receptor for the Ulex europaeus lectin were also observed in the yolk sac vasculature following injection of either unfractionated or CD34+ day -10 hEB cells. Primitive angiohematopoietic stem cells (total and CD34+ day -10 hEB cells) as well as adult hematopoietic stem cells could home to intraembryonic blood-forming organs following injection into the yolk sac. These observations demonstrate the utility of the avian embryo as a convenient and reliable host to model the angiohematopoietic development of human embryonic, or other early stem cells.

Definitive erythropoiesis in chicken yolk sac

The first wave of erythropoiesis in amniotic animals generates all primitive erythrocytes and takes place exclusively in yolk sac mesoderm. It is less clear, however, to what extent and for how long the yolk sac contributes to the second wave of erythropoiesis which gives rise to definitive erythrocytes for later embryonic and adult use. Here, we examine the initiation, duration, and site of definitive erythrocyte formation in chicken yolk sac. We show that the earliest definitive erythrocytes are generated in yolk sac venous vessels surrounding major arteries at embryonic day (E) 4-4.5, and that mature definitive erythrocytes enter circulating at E4.5-E5. This takes place at a time when yolk sac vasculature remodels extensively to generate paired arterial/venous vessels. The yolk sac remains the predominant site for definitive erythropoiesis from E5 to E10, and continues to generate definitive erythrocytes at least until E15. Similar to primitive erythropoiesis, definitive erythropoiesis in the yolk sac is accompanied by the expression of transcriptional regulators gata1, scl, and lmo2. Furthermore, our data suggest that one main source of definitive erythropoietic cells is the pre-existing vascular endothelial cells. It remains unclear whether yolk sac derived hematopoietic progenitors that do not undergo erythropoiesis in the yolk sac may take up intraembryonic niches and contribute to erythropoietic stem cell population after hatching.

Cardiovascular and Hematopoietic Defects Associated With Notch1 Activation in Embryonic Tie2-Expressing Populations

Notch signaling is critical for the development and maintenance of the cardiovasculature, with loss-of-function studies defining roles of Notch1 in the endothelial/hematopoietic lineages. No in vivo studies have addressed complementary gain-of-function strategies within these tissues to define consequences of Notch activation. We developed a transgenic model of Cre recombinase-mediated activation of a constitutively active mouse Notch1 allele (N1ICD(+)) and studied transgene activation in Tie2-expressing lineages. The in vivo phenotype was compared to effects of Notch1 activation on endothelial tubulogenesis, paracrine regulation of smooth muscle cell proliferation, and hematopoiesis. N1ICD(+) embryos showed midgestation lethality with defects in angiogenic remodeling of embryonic and yolk sac vasculature, cardiac development, smooth muscle cell investment of vessels, and hematopoietic differentiation. Angiogenic defects corresponded with impaired endothelial tubulogenesis in vitro following Notch1 activation and paracrine inhibition of smooth muscle cells when grown with Notch1-activated endothelial cells. Flow cytometric analysis of hematopoietic and endothelial precursor populations demonstrated a significant loss of CD71(+)/Ter119(+) populations with an active N1ICD(+) allele and a corresponding increase in c-Kit(+)/CD71 and Flk1(+) populations, suggesting a developmental block during the transition between c-Kit- and Ter119-expressing erythroblasts. Cardiovascular lineages are sensitive to an imbalance in Notch signaling, with aberrant activation reflecting a vascular phenotype comparable to a loss-of-function Notch1 mutation.

Redundant Roles of Tead1 and Tead2 in Notochord Development and the Regulation of Cell Proliferation and Survival

Four members of the TEAD/TEF family of transcription factors are expressed widely in mouse embryos and adult tissues. Although in vitro studies have suggested various roles for TEAD proteins, their in vivo functions remain poorly understood. Here we examined the role of Tead genes by generating mouse mutants for Tead1 and Tead2. Tead2(-/-) mice appeared normal, but Tead1(-/-); Tead2(-/-) embryos died at embryonic day 9.5 (E9.5) with severe growth defects and morphological abnormalities. At E8.5, Tead1(-/-); Tead2(-/-) embryos were already small and lacked characteristic structures such as a closed neural tube, a notochord, and somites. Despite these overt abnormalities, differentiation and patterning of the neural plate and endoderm were relatively normal. In contrast, the paraxial mesoderm and lateral plate mesoderm were displaced laterally, and a differentiated notochord was not maintained. These abnormalities and defects in yolk sac vasculature organization resemble those of mutants for Yap, which encodes a coactivator of TEAD proteins. Moreover, we demonstrated genetic interactions between Tead1 and Tead2 and Yap. Finally, Tead1(-/-); Tead2(-/-) embryos showed reduced cell proliferation and increased apoptosis. These results suggest that Tead1 and Tead2 are functionally redundant, use YAP as a major coactivator, and support notochord maintenance as well as cell proliferation and survival in mouse development.

Blockade of c-Jun N-terminal kinase activation abrogates hyperglycemia-induced yolk sac vasculopathy in vitro

Maternal hyperglycemia has an impact on both the function and morphology of the rodent visceral yolk sac; the objective of the present study was to determine whether hyperglycemia in vitro affects yolk sac vasculogenesis. Because maternal hyperglycemia triggers c-Jun N-terminal kinase (JNK) 1 and 2 activation in the yolk sac, we tested whether the inhibition of JNK activation would ameliorate hyperglycemia-induced yolk sac vasculopathy. In rodents, the yolk sac serves as the primitive placenta after implantation and before the formation of the chorioallantoic placenta. Furthermore, during this early stage, the nutrition from mother to embryo is considered to be facilitated by a tissue-to-tissue form of nutrition, referred to as histiotropic nutrition, and subsequently via yolk sac facilitation (hemotropic nutrition). In addition, during embryopathy, teratogen such as hyperglycemia is associated with concomitant injury to the yolk sac and embryo. Rat embryos at embryonic day 9 were cultured under euglycemic (150 mg/dL glucose) and hyperglycemic (500 mg /dL glucose) conditions. JNK activation was inhibited using a JNK1/2-specific inhibitor SP60025 at concentrations of 40, 400, and 800 nM. After 48 hours, the development of yolk sac vasculatures was evaluated by assigning to arbitrative scores on the basis of yolk sac vasculature morphology. The correlation between yolk sac vasculature and embryonic malformation rates was assessed. Levels of phosphorylated JNK1/2 and Bcl-2-associated X protein (Bax) in the yolk sacs from conceptuses of the euglycemic and hyperglycemic groups were determined by Western blotting with densitometric quantification. Under hyperglycemic conditions, yolk sac development was morphologically impaired. The yolk sac vasculature score of the hyperglycemic group was significantly lower than that of the euglycemic group. Yolk sac vasculature morphologic scores were inversely correlated with embryonic malformation rates. Levels of phosphorylated JNK1/2 and Bax in yolk sacs of the hyperglycemic group were significantly higher than those in yolk sacs of the euglycemic group. JNK1/2-specific inhibitor, SP600125, ameliorated the adverse effect of hyperglycemia on yolk sac vasculature development. Whereas the vasculature morphologic score of yolk sacs in the hyperglycemic group was 54% lower than that of euglycemic group, the vasculature morphologic score of yolk sacs in hyperglycemic plus 800 nM SP600125 group was as same as that in the euglycemic group. Thus, SP600125 at 800 nM completely reversed hyperglycemia-induced vasculopathy as well as embryopathy. Hyperglycemia in vitro induces yolk sac vasculopathy. Embryonic malformation is inversely correlated with the yolk sac vasculature development, suggesting that hyperglycemia-induced yolk sac vasculopathy may be one of the causative factors in hyperglycemia-induced embryonic malformation. Blockade of JNK1/2 activation restores hyperglycemia-induced vasculopathy and reduces the malformation rates. These findings indicate that JNK1/2 activation mediates the deleterious effect of hyperglycemia on yolk sac vasculature and embryonic development.

CARDIAC TISSUE FACTOR: ROLES IN PHYSIOLOGY AND FIBROSIS

1. The aim of the present review is to discuss: (i) the role of tissue factor (TF) in the heart and focus on its potential role in maintaining normal extracellular cardiac homeostasis; (ii) the means by which TF may be contributing to extracellular matrix regulation and vascular maintenance; and (iii) potential mechanisms whereby reduced TF expression leads to cardiac fibrosis. 2. Tissue factor is the main initiator of the coagulation cascade in response to tissue injury, but it is also involved in numerous other biological processes, including angiogenesis, cell migration, apoptosis, metastasis and inflammation. 3. Tissue factor is implicated in cardiovascular disease and its localization and level of expression in cardiomyocytes suggests a unique role in maintaining the structure and function of cardiac muscle. 4. It has been shown that TF(-/-) mice die in utero as a result of disrupted yolk sac vasculature. Low-TF mice, which have transgenic expression of TF at less than 1% of normal levels, are rescued from lethality. 5. Low-TF mice develop cardiac fibrosis in a gender-dependent manner that may be dependent on differential expression of urokinase plasminogen activator. 6. Intracardiac bleeding in low-TF mice may occur as a result of a primary haemostasis defect and/or as a result of disrupted vascular maintenance. The mechanism, when elucidated, will have important therapeutic implications and may provide novel strategies for the treatment of cardiac fibrosis.

Compensatory signalling induced in the yolk sac vasculature by deletion of TGFβ receptors in mice

Vascular development depends on transforming growth factor beta (TGFbeta), but whether signalling of this protein is required for the development of endothelial cells (ECs), vascular smooth muscle cells (VSMCs) or both is unclear. To address this, we selectively deleted the type I (ALK5, TGFBR1) and type II (TbetaRII, TGFBR2) receptors in mice. Absence of either receptor in ECs resulted in vascular defects in the yolk sac, as seen in mice lacking receptors in all cells, causing embryonic lethality at embryonic day (E)10.5. Deletion of TbetaRII specifically in VSMCs also resulted in vascular defects in the yolk sac; however, these were observed at later stages of development, allowing the embryo to survive to E12.5. Because TGFbeta can also signal in ECs via ALK1 (ACVRL1), we replaced ALK5 by a mutant defective in SMAD2 and SMAD3 (SMAD2/3) activation that retained the ability to transactivate ALK1. This again caused defects in the yolk sac vasculature with embryonic lethality at E10.5, demonstrating that TGFbeta/ALK1 signalling in ECs cannot compensate for the lack of TGFbeta/ALK5-induced SMAD2/3 signalling in vivo. Unexpectedly, SMAD2 phosphorylation and alpha-smooth muscle actin (SMAalpha, ACTA2) expression occurred in the yolk sacs of ALK5(-/-) embryos and ALK5(-/-) embryonic stem cells undergoing vasculogenesis, and these processes could be blocked by an ALK4 (ACVR1B)/ALK5 inhibitor. Together, the data show that ALK5 is required in ECs and VSMCs for yolk sac vasculogenesis; in the absence of ALK5, ALK4 mediates SMAD2 phosphorylation and consequently SMAalpha expression.

Multiple cardiovascular defects caused by the absence of alternatively spliced segments of fibronectin

Alternatively spliced variants of fibronectin (FN) containing exons EIIIA and EIIIB are expressed around newly forming vessels in development and disease but are downregulated in mature vasculature. The sequences and patterns of expression of these splice variants are highly conserved among vertebrates, suggestive of their biological importance; however the functions of EIIIA and EIIIB-containing FNs are unknown. To understand the role(s) of these splice variants, we deleted both EIIIA and EIIIB exons from the FN gene and observed embryonic lethality with incomplete penetrance by embryonic day 10.5. Deletion of both EIIIA and EIIIB exons did not affect synthesis or cell surface deposition of FN, indicating that embryonic lethality was due specifically to the absence of EIIIA and EIIIB exons from FN. EIIIA/EIIIB double-null embryos displayed multiple embryonic cardiovascular defects, including vascular hemorrhage, failure of remodeling embryonic and yolk sac vasculature, defective placental angiogenesis and heart defects. In addition, we observed defects in coverage and association with dorsal aortae of alpha-smooth-muscle-actin-positive cells. Our studies indicate that the presence or absence of EIIIA and EIIIB exons alters the function of FN and demonstrate the requirement for these alternatively spliced exons in cardiovascular development.

Cell tracing shows the contribution of the yolk sac to adult haematopoiesis

The first haematopoietic stem cells (HSCs) appear in the aorta-gonad-mesonephros (AGM) region, major vitelline and umbilical vessels, and placenta; however, whether they arise locally or from immigrant yolk sac precursor cells remains unclear. This issue is best addressed by measuring cell-lineage relationships rather than cell potentials. To undertake long-term in vivo tracing of yolk sac cells, we designed a non-invasive pulse-labelling system based on Cre/loxP recombination. Here we show that in Runx1(+/-) (runt-related transcription factor 1) heterozygous mice, yolk sac cells expressing Runx1 at embryonic day 7.5 develop into fetal lymphoid progenitors and adult HSCs. During mid-gestation the labelled (embryonic day 7.5) yolk sac cells colonize the umbilical cord, the AGM region and subsequently the embryonic liver. This raises the possibility that some HSCs associated with major embryonic vasculature are derived from yolk sac precursors. We observed virtually no contribution of the labelled cells towards the yolk sac vasculature, indicating early segregation of endothelial and haematopoietic lineages.

Maturation and enucleation of primitive erythroblasts during mouse embryogenesis is accompanied by changes in cell-surface antigen expression

AbstractPrimitive erythroblasts (EryPs) are the first hematopoietic cell type to form during mammalian embryogenesis and emerge within the blood islands of the yolk sac. Large, nucleated EryPs begin to circulate around midgestation, when connections between yolk sac and embryonic vasculature mature. Two to 3 days later, small cells of the definitive erythroid lineage (EryD) begin to differentiate within the fetal liver and rapidly outnumber EryPs in the circulation. The development and maturation of EryPs remain poorly defined. Our analysis of embryonic blood at different stages reveals a stepwise developmental progression within the EryP lineage from E9.5 to E12.5. Thereafter, EryDs are also present in the bloodstream, and the 2 lineages are not easily distinguished. We have generated a transgenic mouse line in which the human ϵ-globin gene promoter drives expression of green fluorescent protein exclusively within the EryP lineage. Here, we have used this line to characterize changes in cell morphology and surface-marker expression as EryPs mature and to track EryP numbers and enucleation throughout gestation. This study identifies previously unrecognized synchronous developmental stages leading to the maturation of EryPs in the mouse embryo. Unexpectedly, we find that EryPs are a stable cell population that persists through the end of gestation.

A pivotal role for endogenous TGF-β-activated kinase-1 in the LKB1/AMP-activated protein kinase energy-sensor pathway

TGF-beta-activated kinase-1 (TAK1), also known as MAPKK kinase-7 (MAP3K7), is a candidate effector of multiple circuits in cardiac biology and disease. Here, we show that inhibition of TAK1 in mice by a cardiac-specific dominant-negative mutation evokes electrophysiological and biochemical properties reminiscent of human Wolff-Parkinson-White syndrome, arising from mutations in AMP-activated protein kinase (AMPK), most notably, accelerated atrioventricular conduction and impaired AMPK activation. To test conclusively the biochemical connection from TAK1 to AMPK suggested by this phenotype, we disrupted TAK1 in mouse embryos and embryonic fibroblasts by Cre-mediated recombination. In TAK1-null embryos, the activating phosphorylation of AMPK at T172 was blocked, accompanied by defective AMPK activity. However, loss of endogenous TAK1 causes midgestation lethality, with defective yolk sac and intraembryonic vasculature. To preclude confounding lethal defects, we acutely ablated floxed TAK1 in culture by viral delivery of Cre. In culture, endogenous TAK1 was activated by oligomycin, the antidiabetic drug metformin, 5-aminoimidazole-4-carboxamide riboside (AICAR), and ischemia, well established triggers of AMPK activity. Loss of TAK1 in culture blocked T172 phosphorylation induced by all three agents, interfered with AMPK activation, impaired phosphorylation of the endogenous AMPK substrate acetyl CoA carboxylase, and also interfered with activation of the AMPK kinase LKB1. Thus, by disrupting the endogenous TAK1 locus, we prove a pivotal role for TAK1 in the LKB1/AMPK signaling axis, an essential governor of cell metabolism.

Autotaxin Stabilizes Blood Vessels and Is Required for Embryonic Vasculature by Producing Lysophosphatidic Acid

Autotaxin (ATX) is a cancer-associated motogen that has multiple biological activities in vitro through the production of bioactive small lipids, lysophosphatidic acid (LPA). ATX and LPA are abundantly present in circulating blood. However, their roles in circulation remain to be solved. To uncover the physiological role of ATX we analyzed ATX knock-out mice. In ATX-null embryos, early blood vessels appeared to form properly, but they failed to develop into mature vessels. As a result ATX-null mice are lethal around embryonic day 10.5. The phenotype is much more severe than those of LPA receptor knock-out mice reported so far. In cultured allantois explants, neither ATX nor LPA was angiogenic. However, both of them helped to maintain preformed vessels by preventing disassembly of the vessels that was not antagonized by Ki16425, an LPA receptor antagonist. In serum from heterozygous mice both lysophospholipase D activity and LPA level were about half of those from wild-type mice, showing that ATX is responsible for the bulk of LPA production in serum. The present study revealed a previously unassigned role of ATX in stabilizing vessels through novel LPA signaling pathways.

Ablation of Mouse Phosphomannose Isomerase (Mpi) Causes Mannose 6-Phosphate Accumulation, Toxicity, and Embryonic Lethality

MPI encodes phosphomannose isomerase, which interconverts fructose 6-phosphate and mannose 6-phosphate (Man-6-P), used for glycoconjugate biosynthesis. MPI mutations in humans impair protein glycosylation causing congenital disorder of glycosylation Ib (CDG-Ib), but oral mannose supplements normalize glycosylation. To establish a mannose-responsive mouse model for CDG-Ib, we ablated Mpi and provided dams with mannose to rescue the anticipated defective glycosylation. Surprisingly, although glycosylation was normal, Mpi(-/-) embryos died around E11.5. Mannose supplementation even hastened their death, suggesting that man-nose was toxic. Mpi(-/-) embryos showed growth retardation and placental hyperplasia. More than 90% of Mpi(-/-) embryos failed to form yolk sac vasculature, and 35% failed chorioallantoic fusion. We generated primary embryonic fibroblasts to investigate the mechanisms leading to embryonic lethality and found that mannose caused a concentration- and time-dependent accumulation of Man 6-P in Mpi(-/-) fibroblasts. In parallel, ATP decreased by more than 70% after 24 h compared with Mpi(+/+) controls. In cell lysates, Man-6-P inhibited hexokinase (70%), phosphoglucose isomerase (65%), and glucose-6-phosphate dehydrogenase (85%), but not phosphofructokinase. Incubating intact Mpi(-/-) fibroblasts with 2-[(3)H]deoxyglucose confirmed mannose-dependent hexokinase inhibition. Our results in vitro suggest that mannose toxicity in Mpi(-/-) embryos is caused by Man-6-P accumulation, which inhibits glucose metabolism and depletes intracellular ATP. This was confirmed in E10.5 Mpi(-/-) embryos where Man-6-P increased more than 10 times, and ATP decreased by 50% compared with Mpi(+/+) littermates. Because Mpi ablation is embryonic lethal, a murine CDG-Ib model will require hypomorphic Mpi alleles.