Yolk Sac Blood Vessels Research Articles

Adult-repopulating lymphoid potential of yolk sac blood vessels is not confined to arterial endothelial cells.

During embryogenesis, hematopoietic stem progenitor cells (HSPCs) are believed to be derived from hemogenic endothelial cells (HECs). Moreover, arterial feature is proposed to be a prerequisite for HECs to generate HSPCs with lymphoid potential. Although the molecular basis of hematopoietic stem cell-competent HECs has been delicately elucidated within the embryo proper, the functional and molecular characteristics of HECs in the extraembryonic yolk sac (YS) remain largely unresolved. In this study, we initially identified six molecularly different endothelial populations in the midgestational YS through integrated analysis of several single-cell RNA sequencing (scRNA-seq) datasets and validated the arterial vasculature distribution of Gja5+ ECs using a Gja5-EGFP reporter mouse model. Further, we explored the hemogenic potential of different EC populations based on their Gja5-EGFP and CD44 expression levels. The hemogenic potential was ubiquitously detected in spatiotemporally different vascular beds on embryonic days (E)8.5-E9.5 and gradually concentrated in CD44-positive ECs from E10.0. Unexpectedly, B-lymphoid potential was detected in the YS ECs as early as E8.5 regardless of their arterial features. Furthermore, the capacity for generating hematopoietic progenitors with in vivo lymphoid potential was found in nonarterial as well as arterial YS ECs on E10.0-E10.5. Importantly, the distinct identities of E10.0-E10.5 HECs between YS and intraembryonic caudal region were revealed by further scRNA-seq analysis. Cumulatively, these findings extend our knowledge regarding the hemogenic potential of ECs from anatomically and molecularly different vascular beds, providing a theoretical basis for better understanding the sources of HSPCs during mammalian development.

Cannibalized erythroblasts accelerate developmental neurogenesis by regulating mitochondrial dynamics

Metabolic support was long considered to be the only developmental function of hematopoiesis, a view that is gradually changing. Here, we disclose a mechanism triggered during neurulation that programs brain development by donation of sacrificial yolk sac erythroblasts to neuroepithelial cells. At embryonic day (E) 8.5, neuroepithelial cells transiently integrate with the endothelium of yolk sac blood vessels and cannibalize intravascular erythroblasts as transient heme-rich endosymbionts. This cannibalistic behavior instructs precocious neuronal differentiation of neuroepithelial cells in the proximity of blood vessels. By experiments invitro, we show that access to erythroblastic heme accelerates the pace of neurogenesis by induction of a truncated neurogenic differentiation program from a poised state. Mechanistically, the poised state is invoked by activation of the mitochondrial electron transport chain that leads to amplified production of reactive oxygen species in addition to omnipresent guanosine triphosphate (GTP) with consequential upregulation of pro-differentiation β-catenin.

Breast cancer resistance protein (Bcrp/Abcg2) is selectively modulated by lipopolysaccharide (LPS) in the mouse yolk sac

Bacterial infection alters placental ABC transporters expression. These transporters provide fetal protection against circulating xenobiotics and environmental toxins present in maternal blood. We hypothesized that lipopolysaccharide (LPS-bacterial mimic) alters the yolk sac morphology and expression of key ABC transporters in a gestational-age dependent manner. Yolk sac samples from C57BL/6 mice were obtained at gestational ages (GD) 15.5 and GD18.5, 4 or 24 h after LPS exposure (150ug/kg; n = 8/group). Samples underwent morphometrical, qPCR and immunohistochemistry analysis. The volumetric proportions of the histological components of the yolk sac did not change in response to LPS. LPS increased Abcg2 expression at GD15.5, after 4 h of treatment (p < 0.05). No changes in Abca1, Abcb1a/b, Abcg1, Glut1, Snat1, Il-1β, Ccl2 and Mif were observed. Il-6 and Cxcl1 were undetectable in the yolk sac throughout pregnancy. Abca1, breast cancer resistance protein (Bcrp, encoded by Abcg2) and P-glycoprotein (P-gp/ Abcb1a/b) were localized in the endodermal (uterine-facing) epithelium and to a lesser extent in the mesothelium (amnion-facing), whereas Abca1 was also localized to the endothelium of the yolk sac blood vessels. LPS increased the labeling area and intensity of Bcrp in the yolk sac’s mesothelial cells at GD15.5 (4 h), whereas at GD18.5, the area of Bcrp labeling in the mesothelium (4 and 24 h) was decreased (p < 0.05). Bacterial infection has the potential to change yolk sac barrier function by affecting Bcrp and Abcg2 expression in a gestational-age dependent-manner. These changes may alter fetal exposure to xenobiotics and toxic substances present in the maternal circulation and in the uterine cavity.

Yolk sac development in lizards (Lacertilia: Scincidae): New perspectives on the egg of amniotes.

Embryos of oviparous reptiles develop on the surface of a large mass of yolk, which they metabolize to become relatively large hatchlings. Access to the yolk is provided by tissues growing outward from the embryo to cover the surface of the yolk. A key feature of yolk sac development is a dedicated blood vascular system to communicate with the embryo. The best known model for yolk sac development and function of oviparous amniotes is based on numerous studies of birds, primarily domestic chickens. In this model, the vascular yolk sac forms the perimeter of the large yolk mass and is lined by a specialized epithelium, which takes up, processes and transports yolk nutrients to the yolk sac blood vessels. Studies of lizard yolk sac development, dating to more than 100 years ago, report characteristics inconsistent with this model. We compared development of the yolk sac from oviposition to near hatching in embryonic series of three species of oviparous scincid lizards to consider congruence with the pattern described for birds. Our findings reinforce results of prior studies indicating that squamate reptiles mobilize and metabolize the large yolk reserves in their eggs through a process unknown in other amniotes. Development of the yolk sac of lizards differs from birds in four primary characteristics, migration of mesoderm, proliferation of endoderm, vascular development and cellular diversity within the yolk sac cavity. Notably, all of the yolk is incorporated into cells relatively early in development and endodermal cells within the yolk sac cavity align along blood vessels which course throughout the yolk sac cavity. The pattern of uptake of yolk by endodermal cells indicates that the mechanism of yolk metabolism differs between lizards and birds and that the evolution of a fundamental characteristic of embryonic nutrition diverged in these two lineages. Attributes of the yolk sac of squamates reveal the existence of phylogenetic diversity among amniote lineages and raise new questions concerning the evolution of the amniotic egg. J. Morphol. 278:574-591, 2017. © 2017 Wiley Periodicals, Inc.

Pias1 is essential for erythroid and vascular development in the mouse embryo

The protein inhibitor of activated STAT-1 (PIAS1) is one of the few known SUMO E3 ligases. PIAS1 has been implicated in several biological processes including repression of innate immunity and DNA repair. However, PIAS1 function during development and tissue differentiation has not been studied. Here, we report that Pias1 is required for proper embryonic development. Approximately 90% of Pias1 null embryos die in utero between E10.5 and E12.5. We found significant apoptosis within the yolk sac (YS) blood vessels and concomitant loss of red blood cells (RBCs) resulting in profound anemia. In addition, Pias1 loss impairs YS angiogenesis and results in defective capillary plexus formation and blood vessel occlusions. Moreover, heart development is impaired as a result of loss of myocardium muscle mass. Accordingly, we found that Pias1 expression in primary myoblasts enhances the induction of cardiac muscle genes MyoD, Myogenin and Myomaker. PIAS1 protein regulation of cardiac gene transcription is dependent on transcription factors Myocardin and Gata-4. Finally, endothelial cell specific inactivation of Pias1 in vivo impairs YS erythrogenesis, angiogenesis and recapitulates loss of myocardium muscle mass. However, these defects are not sufficient to recapitulate the lethal phenotype of Pias1 null embryos. These findings highlight Pias1 as an essential gene for YS erythropoiesis and vasculogenesis in vivo.

Tie‐2‐Cre‐mediated Inactivation of Lipid phosphate phosphatase (Lpp)‐3 Results in Vascular Defects

RationaleMouse embryos with null Lpp3 alleles die as early as E6.5days as they fail to form chorio‐allantoic placenta and yolk‐sac blood vessels. Thus, in vivo function of Lpp3 gene during later stages of embryonic development is not known.GoalTo examine tissue specific function of Lpp3, we generated Lpp3flox/flox mice. To delete Lpp3 in endothelial cell (ECs), we crossed Lpp3flox/flox mice with Tie‐2‐Cre transgenic line.ResultsIn contrast to the conventional Lpp3 deletion, Tie‐2‐Cre mediated Lpp3 deletion in ECs did not affect normal vasculogenesis; however, this led to impaired angiogenesis and late embryonic lethal phenotype. Microscopic analyses revealed reduced fibronectin and VE‐cadherin expression, and increased apoptosis in CD31‐positive vascular structures. Transmission electron microscopy confirmed the presence of apoptotic ECs in mutants. Accordingly, Lpp3 null cells showed increase in p53 and p21 levels, with concomitant decrease in cell proliferation as illustrated by reduced 6‐Bromodeoxyuridine incorporation. In vitro, LPP3 knockdown decreased transendothelial electrical resistance, interestingly re‐expression of β‐catenin cDNA into LPP3 depleted ECs partially restored the effect of loss of LPP3.ConclusionThese results define a critical role of LPP3 in angiogenesis, vascular development and control of apoptotic pathway.Support: NIH R01HL07935, UIC CCTS UL1RR029879.

The Chromatin-Remodeling Enzymes BRG1 and CHD4 Antagonistically Regulate Vascular Wnt Signaling

Canonical Wnt signaling plays an important role in embryonic and postnatal blood vessel development. We previously reported that the chromatin-remodeling enzyme BRG1 promotes vascular Wnt signaling. Vascular deletion of Brg1 results in aberrant yolk sac blood vessel morphology, which is rescued by pharmacological stimulation of Wnt signaling with lithium chloride (LiCl). We have now generated embryos lacking the chromatin-remodeling enzyme Chd4 in vascular endothelial cells. Unlike Brg1 mutants, Chd4 mutant embryos had normal yolk sac vascular morphology. However, concomitant deletion of Chd4 and Brg1 rescued vascular abnormalities seen in Brg1 mutant yolk sacs to the same extent as LiCl treatment. We hypothesized that Wnt signaling was upregulated in Chd4 mutant yolk sac vasculature. Indeed, we found that Chd4 deletion resulted in upregulation of the Wnt-responsive transcription factor Tcf7 and an increase in Wnt target gene expression in endothelial cells. Furthermore, we identified one Wnt target gene, Pitx2, that was downregulated in Brg1 mutant endothelial cells but was rescued following LiCl treatment and in Brg1 Chd4 double mutant vasculature, suggesting that PITX2 helps to mediate the restoration of yolk sac vascular remodeling under both conditions. We conclude that BRG1 and CHD4 antagonistically modulate Wnt signaling in developing yolk sac vessels to mediate normal vascular remodeling.

Protective effects of β‐carotene and melanin against protoporphyrine IX‐induced phototoxicity in the photo hen's egg test

The aim of our study was to evaluate the photoprotective potential of melanin and β-carotene against protoporphyrine IX-induced phototoxicity via photo hen's egg test. In three independent test groups, the yolk sac blood vessel system of hen's eggs was exposed to protoporphyrine IX and irradiated with ultraviolet A (UVA). One of the test groups also received melanin to investigate its photoprotective capacity; another test group received β-carotene for the same purpose. Morphological changes and embryo lethality were recorded in these three test groups for a period of 24 h. The same parameters were obtained in five different control groups. The control groups exhibited only minimal morphological changes and no fatalities. In contrast, severe phototoxic damage and a high lethality rate (75%) were observed in the test group exposed to protoporphyrine IX and UVA. Lethality was somewhat lower in the β-carotene test group (58%) and was considerably lower in the melanin test group (17%). The photoprotective potential against protoporphyrine IX-induced phototoxic damage was moderate for β-carotene and was remarkable for melanin. Given that synthetic melanocyte stimulating hormone (MSH) analogues induce a de novo synthesis of melanin without any previous ultraviolet irradiation in human skin, the application of MSH analogues might be conceived of as 'light hardening' without light. Synthetic MSH analogues thus may represent a new promising therapeutic option for photodermatoses especially for erythropoietic protoporphyria.

Separating genetic and hemodynamic defects in neuropilin 1

Formation of the cardiovascular system is crucial for embryo growth and survival and the importance of proper blood flow even during these early stages is becoming increasingly clear. In recent years, there has been a debate regarding which aspects of early vascular formation are genetically predetermined, and which are governed by external stimuli, such as mechanical and biochemical signaling. Though the initial vascular network is patterned without knowledge of the functional requirements, it makes biological sense that a feedback mechanism with respect to physiological function would set in as soon as this information becomes available. We recently showed that physical forces created by blood flow are necessary for proper vascular development and remodeling. In this study, we show how to distinguish between genetic defects and biomechanical defects during vascular development. Using mouse embryo culture, we study hemodynamic defects in Nrp1−/− mice together with an analysis of the emerging vascular defects. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow‐induced deficiencies from those caused by the nrp‐1 mutation, we arrested blood flow in cultured wild‐type and mutant embryos and followed their vascular development. This method allows genetic defects caused by loss‐of‐function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.Grant Funding SourceNSERC

Platelet-derived growth factor receptors direct vascular development independent of vascular smooth muscle cell function.

Complete loss of platelet-derived growth factor (PDGF) receptor signaling results in embryonic lethality around embryonic day 9.5, but the cause of this lethality has not been identified. Because cardiovascular failure often results in embryonic lethality at this time point, we hypothesized that a failure in cardiovascular development could be the cause. To assess the combined role of PDGF receptor alpha (PDGFRalpha) and PDGFRbeta, we generated embryos that lacked these receptors in cardiomyocytes and vascular smooth muscle cells (VSMC) using conditional gene ablation. Deletion of either PDGFRalpha or PDGFRbeta caused no overt vascular defects, but loss of both receptors using an SM22alpha-Cre transgenic mouse line led to a disruption in yolk sac blood vessel development. The cell population responsible for this vascular defect was the yolk sac mesothelial cells, not the cardiomyocytes or the VSMC. Coincident with loss of PDGF receptor signaling, we found a reduction in collagen deposition and an increase in MMP-2 activity. Finally, in vitro allantois cultures demonstrated a requirement for PDGF signaling in vessel growth. Together, these data demonstrate that PDGF receptors cooperate in the yolk sac mesothelium to direct blood vessel maturation and suggest that these effects are independent of their role in VSMC development.

Separating genetic and hemodynamic defects in neuropilin 1 knockout embryos

Targeted inactivation of genes involved in murine cardiovascular development frequently leads to abnormalities in blood flow. As blood fluid dynamics play a crucial role in shaping vessel morphology, the presence of flow defects generally prohibits the precise assignment of the role of the mutated gene product in the vasculature. In this study, we show how to distinguish between genetic defects caused by targeted inactivation of the neuropilin 1 (Nrp1) receptor and hemodynamic defects occurring in homozygous knockout embryos. Our analysis of a Nrp1 null allele bred onto a C57BL/6 background shows that vessel remodeling defects occur concomitantly with the onset of blood flow and cause death of homozygous mutants at E10.5. Using mouse embryo culture, we establish that hemodynamic defects are already present at E8.5 and continuous circulation is never established in homozygous mutants. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow-induced deficiencies from those caused by the Nrp1 mutation, we arrested blood flow in cultured wild-type and mutant embryos and followed their vascular development. We find that loss of Nrp1 function rather than flow induces the altered geometry of the capillary plexus. Endothelial cell migration, but not replication, is altered in Nrp1 mutants. Gene expression analysis of endothelial cells isolated from freshly dissected wild-type and mutants and after culture in no-flow conditions showed down-regulation of the arterial marker genes connexin 40 and ephrin B2 related to the loss of Nrp1 function. This method allows genetic defects caused by loss-of-function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.

Separating Genetic and Hemodynamics Effects In Nrp1 Knockout Embryos

Targeted inactivation of genes involved in murine cardiovascular development frequently leads to abnormalities in blood flow. Since blood fluid dynamics play a crucial role in shaping vessel morphology, the presence of flow defects generally prohibits the precise assignment of the role of the mutated gene product in the vasculature. In this study, we show how to distinguish between genetic defects caused by targeted inactivation of the Neuropilin‐1 (Nrp‐1) receptor, and hemodynamic defects occurring in homozygous knockout embryos. Using mouse embryo culture, we establish that hemodynamic defects are already present at E8.5 and continuous circulation is never established in homozygous mutants. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow‐induced deficiencies from those caused by the nrp‐1 mutation, we arrested blood flow in cultured wild‐type and mutant embryos and followed their vascular development. We find that loss of nrp‐1 function rather than flow induces the altered geometry of the capillary plexus. This method allows genetic defects caused by loss‐of‐function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.

Defective vascular morphogenesis and mid-gestation embryonic death in mice lacking RA-GEF-1

A multitude of guanine nucleotide exchange factors (GEFs) regulate Rap1 small GTPases, however, their individual functions remain obscure. Here, we investigate the in vivo function of the Rap1 GEF RA-GEF-1. The expression of RA-GEF-1 in wild-type mice starts at embryonic day (E) 8.5, and continues thereafter. RA-GEF-1 −/− mice appear normal until E7.5, but become grossly abnormal and dead by E9.5. This mid-gestation death appears to be closely associated with severe defects in yolk sac blood vessel formation. RA-GEF-1 −/− yolk sacs form apparently normal blood islands by E8.5, but the blood islands fail to coalesce into a primary vascular plexus, indicating that vasculogenesis is impaired. Furthermore, RA-GEF-1 −/− embryos proper show severe defects in the formation of major blood vessels. These results suggest that deficient Rap1 signaling may lead to defective vascular morphogenesis in the yolk sac and embryos proper.

Phenotypic analysis of a novel chordin mutant in medaka

We have isolated and characterized a ventralized mutant in medaka (the Japanese killifish; Oryzias latipes), which turned out to have a mutation in the chordin gene. The mutant exhibits ventralization of the body axis, malformation of axial bones, over-bifurcation of yolk sac blood vessels, and laterality defects in internal organs. The mutant exhibits variability of phenotypes, depending on the culture temperature, from embryos with a slightly ventralized phenotype to those without any head and trunk structures. Taking advantages of these variable and severe phenotypes, we analyzed the role of Chordin-dependent tissues such as the notochord and Kupffer's vesicle (KV) in the establishment of left-right axis in fish. The results demonstrate that, in the absence of the notochord and KV, the medaka lateral plate mesoderm autonomously and bilaterally expresses spaw gene in a default state.

Targeted disruption of cubilin reveals essential developmental roles in the structure and function of endoderm and in somite formation

BackgroundCubilin is a peripheral membrane protein that interacts with the integral membrane proteins megalin and amnionless to mediate ligand endocytosis by absorptive epithelia such as the extraembryonic visceral endoderm (VE).ResultsHere we report the effects of the genetic deletion of cubilin on mouse embryonic development. Cubilin gene deletion is homozygous embryonic lethal with death occurring between 7.5–13.5 days post coitum (dpc). Cubilin-deficient embryos display developmental retardation and do not advance morphologically beyond the gross appearance of wild-type 8–8.5 dpc embryos. While mesodermal structures such as the allantois and the heart are formed in cubilin mutants, other mesoderm-derived tissues are anomalous or absent. Yolk sac blood islands are formed in cubilin mutants but are unusually large, and the yolk sac blood vessels fail to undergo remodeling. Furthermore, somite formation does not occur in cubilin mutants. Morphological abnormalities of endoderm occur in cubilin mutants and include a stratified epithelium in place of the normally simple columnar VE epithelium and a stratified cuboidal epithelium in place of the normally simple squamous epithelium of the definitive endoderm. Cubilin-deficient VE is also functionally defective, unable to mediate uptake of maternally derived high-density lipoprotein (HDL).ConclusionIn summary, cubilin is required for embryonic development and is essential for the formation of somites, definitive endoderm and VE and for the absorptive function of VE including the process of maternal-embryo transport of HDL.

Cytogenetic and histological features of a human embryo with homogeneous chromosome 8 trisomy

Homogeneous and complete trisomy 8 is extremely rare. With one recent neonatal exception, all reported cases have been mosaic, due to mitotic non-disjunction during early zygotic development. We report a case of chromosome 8 trisomy in a human embryo examined at Carnegie stage 11 (25 days post-fertilization). It presented severe cardiovascular and central nervous system malformations. The unusual bifid heart in this embryo spurred a detailed histological examination, karyotyping of a chorionic villus sample and subsequent FISH on inter-phase nuclei of intra-embryonic sections. Trophoblast cells had a karyotype of 47,XX, +8. Within the embryo proper, FISH demonstrated that the trisomy 8 was homogeneous in embryonic as well as extra-embryonic tissues. FQ-PCR supports a meiosis I origin of non-disjunction. In sections, the pharyngeal arches (including cardiac outflow tract), forebrain, mesonephros and liver were absent. Somites and yolk sac blood vessels were irregularly shaped. We show that homogeneous, intra-embryonic trisomy 8 is compatible with implantation and early human development. Molecular pathways that may be compromised and their impact on organogenesis are discussed.

Phototoxicity of exogenous protoporphyrin IX and delta-aminolevulinic acid in the photo hen's egg test.

Oxygen, appropriate light sources, and special photosensitizers are necessary to induce photochemical damage in tumor cells via photodynamic therapy (PDT) delta-aminolevulinic acid (ALA) is increasingly used in PDT, because topical or systemic administration of ALA induces accumulation of endogenous porphyrins preferentially in neoplastic tissues. Subsequent radiation with light of approximately 630 nm leads to selective damage of tumor cells. PDT should optimally leave peritumoral tissues unaffected, but only few data are reported on the effects and the time course of ALA-induced porphyrins in tumor-free tissues. Therefore, we studied the phototoxic effects of protoporphyrin IX (PP) and ALA-induced porphyrins in a recently established phototoxic model based on tumor-free tissue, the photo hen's egg test (PHET). Employing this test procedure, PP provoked strong phototoxic reactions when irradiated with Ultraviolet A immediately and up to 30 h after substance application. In contrast, ALA induced a significant phototoxic effect only if irradiated 24 h after application. Thus, we observed a delayed phototoxic effect of ALA in tumor-free tissue of the yolk sac (YS) blood vessel system. This delayed phototoxic response 24 h after ALA application is probably caused by endogenously synthesized porphyrins. In contrast, epithelial tumors show a maximum porphyrin accumulation 4-8 h after ALA application whereas in healthy human skin porphyrin synthesis is less intensive but prolonged with maximum levels 24-48 h after ALA application. Thus, ALA induced virtually the same delayed phototoxic effect in the tumor-free YS blood vessel tissue as in healthy human skin. These results show that the PHET is a useful model for the predictive preclinical risk assessment of exogenous or endogenous photosensitizers.

Yolk sac infection in a broiler flock: a case report

A case of yolk sac infection is reported among 500 broiler chicks bought for National Root Crop Research Institute, Umudike, Poultry Project. From the day of receipt the birds were depressed, dull with distended abdomen. The next day and subsequent days the birds started dying, reached its peak on the 12th day with 8 deaths. Result of post mortem done on the dead chicks revealed a septicaemic carcass with the subcutaneous and yolk sac blood vessels enlarged and dilated. The striking feature was an inflamed unabsorbed yolk sac with the yolk abnormal in colour and consistency. Post mortem findings seen in chicks that survived into second, third and forth weeks were inspissated yolk sac which contained deeply pigmented caseous material. Bacteriological isolations revealed the presence of Escherichia coli. The outbreak was presumed to be as a result of Escherichia coli contaminating the hatching eggs and further penetrate the shell to infect the yolk. The source of infection was found to be from the hatchery contaminated with Escherichia coli where infection spread from chick to chick. KEY WORDS: Yolk Sac Infection, Broiler Flocks, Septicaemic Carcass. (Nigerian Veterinary Journal: 2002 23(2): 65-69)

Hedgehog is required for murine yolk sac angiogenesis.

Blood islands, the precursors of yolk sac blood vessels, contain primitive erythrocytes surrounded by a layer of endothelial cells. These structures differentiate from extra-embryonic mesodermal cells that underlie the visceral endoderm. Our previous studies have shown that Indian hedgehog (Ihh) is expressed in the visceral endoderm both in the visceral yolk sac in vivo and in embryonic stem (ES) cell-derived embryoid bodies. Differentiating embryoid bodies form blood islands, providing an in vitro model for studying vasculogenesis and hematopoiesis. A role for Ihh in yolk sac function is suggested by the observation that roughly 50% of Ihh(-/-) mice die at mid-gestation, potentially owing to vascular defects in the yolk sac. To address the nature of the possible vascular defects, we have examined the ability of ES cells deficient for Ihh or smoothened (Smo), which encodes a receptor component essential for all hedgehog signaling, to form blood islands in vitro. Embryoid bodies derived from these cell lines are unable to form blood islands, and express reduced levels of both PECAM1, an endothelial cell marker, and alpha-SMA, a vascular smooth muscle marker. RT-PCR analysis in the Ihh(-/-) lines shows a substantial decrease in the expression of Flk1 and Tal1, markers for the hemangioblast, the precursor of both blood and endothelial cells, as well as Flt1, an angiogenesis marker. To extend these observations, we have examined the phenotypes of embryo yolk sacs deficient for Ihh or SMO: Whereas Ihh(-/-) yolk sacs can form blood vessels, the vessels are fewer in number and smaller, perhaps owing to their inability to undergo vascular remodeling. Smo(-/-) yolk sacs arrest at an earlier stage: the endothelial tubes are packed with hematopoietic cells, and fail to undergo even the limited vascular remodeling observed in the Ihh(-/-) yolk sacs. Our study supports a role for hedgehog signaling in yolk sac angiogenesis.

Effects of purified microcystin-LR and cell extracts of Microcystis strains PCC 7813 and CYA 43 on cardiac function in brown trout ( Salmo trutta ) alevins

Sub-lethal cardiac responses of brown trout alevins (Salmo trutta L.) were determined in response to aqueous extracts of the cyanobacterium Microcystis strains PCC 7813 (microcystins detectable by HPLC) and CYA 43 (no microcystins detectable by HPLC) and to the purified cyanobacterial hepatotoxin, microcystin-LR (MC-LR) at concentrations of 5, 50 and 500 μg microcystin-LR equivalents l−1. Responses were determined using a flow chamber and video camera attached to a low power microscope. Heart rate in brown trout alevins was acutely sensitive to cyanobacterial extracts and significant increases were observed within 15–60 sec of exposure to aqueous extracts, although no change was observed on exposure to purified MC-LR. Stroke volume increased in all treatments at 50 and 500 μg MC-LR equivalents l−1, which may, at least in part, have been due to vasodilation of the yolk-sac blood vessels. Cardiac output increased significantly at all three concentrations of cyanobacterial cell extracts but not at the lowest concentration of MC-LR, although the rate increased at levels at/or above 50 μg l−1. Increased heart rate, stroke volume and cardiac output occurred at environmentally relevant microcystin concentrations of Microcystis PCC 7813 and CYA 43 aqueous extracts.