An evaluation, using the comet assay and the micronucleus test, of the antigenotoxic effects of chlorophyll b in mice

We investigated the effects of the dietary pigment chlorophyll b (CLb) on cisplatin (cDDP)-induced oxidative stress and DNA damage, using the comet assay in mouse peripheral blood cells and the micronucleus (MN) test in bone marrow and peripheral blood cells. We also tested for thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) in liver and kidney tissues, as well as catalase (CAT) activity and GSH in total blood. CLb (0.2 and 0.5 mg/kg b.w.) was administrated by gavage every day for 13 days. On the 14th day of the experiment, 6 mg/kg cDDP or saline was delivered intraperitoneally. Treatment with cDDP led to a significant decrease in DNA migration and an increase in MN frequency in both cell types, bone marrow and peripheral blood cells. In the kidneys of mice treated with cDDP, TBARS levels were increased, whereas GSH levels were depleted in kidney and liver. In mice that were pretreated with CLb and then treated with cDDP, TBARS levels maintained normal concentrations and GSH did not differ from cDDP group. The improvement of oxidative stress biomarkers after CLb pre-treatment was associated with a decrease in DNA damage, mainly for the highest dose evaluated. Furthermore, CLb also slightly reduced the frequency of chromosomal breakage and micronucleus formation in mouse bone marrow and peripheral blood cells. These results show that pre-treatment with CLb attenuates cDDP-induced oxidative stress, chromosome instability, and lipid peroxidation. (C) 2011 Elsevier B.V. All rights reserved.

Mechanical vibration preserves bone structure in rats treated with glucocorticoids

Glucocorticoids are an important cause of secondary osteoporosis in humans, which decreases bone quality and leads to fractures. Mechanical stimulation in the form of low-intensity and high-frequency vibration seems to be able to prevent bone loss and to stimulate bone formation. The objective of this study was to evaluate the effects of mechanical vibration on bone structure in rats treated with glucocorticoids. Thirty 3-month-old adult male Wistar rats were randomized to three groups: control (C), glucocorticoid (G), and glucocorticoid with vibration (CV). The G and GV groups received 3.5 mg/kg/day of methylprednisolone 5 days/week for a duration of 9 weeks, and the C group received vehicle (saline solution) during the same period. The CV group was vibrated on a special platform for 30 min per day, 5 days per week during the experiment. The platform was set to provide a vertical acceleration of 1 G and a frequency of 60 Hz. Skeletal bone mass was evaluated by total body densitometry (DXA). Fracture load threshold, undecalcified bone histomorphometry, and bone volume were measured in tibias. Glucocorticoids induced a significantly lower weight gain (-9.7%) and reduced the bone mineral content (-9.2%) and trabecular number (-41.8%) and increased the trabecular spacing (+98.0%) in the G group, when compared to the control (C). Vibration (CV) was able to significantly preserve (29.2%) of the trabecular number and decrease the trabecular spacing (+ 26.6%) compared to the G group, although these parameters did not reach C group values. The fracture load threshold was not different between groups, but vibration significantly augmented the bone volume of the tibia by 21.4% in the CV group compared to the C group. Our study demonstrated that low-intensity and high-frequency mechanical vibration was able to partially inhibit the deleterious consequences of glucocorticoids on bone structure in rats. (C) 2010 Elsevier Inc. All rights reserved.

Immune response to vaccination with DNA-hsp65 in a phase I clinical trial with head and neck cancer patients

DNA-hsp65, a DNA vaccine encoding the 65-kDa heat-shock protein of Mycobacterium leprae (Hsp65) is capable of inducing the reduction of established tumors in mouse models. We conducted a phase I clinical trial of DNA-hsp65 in patients with advanced head and neck carcinoma. In this article, we report on the vaccine`s potential to induce immune responses to Hsp65 and to its human homologue, Hsp60, in these patients. Twenty-one patients with unresectable squamous cell carcinoma of the head and neck received three doses of 150, 400 or 600 mu g naked DNA-hsp65 plasmid by ultrasound-guided intratumoral injection. Vaccination did not increase levels of circulating anti-hsp65 IgG or IgM antibody, or lead to detectable Hsp65-specific cell proliferation or interferon-gamma (IFN-gamma) production by blood mononuclear cells. Frequency of antigen-induced IL-10-producing cells increased after vaccination in 4 of 13 patients analyzed. Five patients showed disease stability or regression following immunization; however, we were unable to detect significant differences between these patients and those with disease progression using these parameters. There was also no increase in antibody or IFN-gamma responses to human Hsp60 in these patients. Our results suggest that although DNA-hsp65 was able to induce some degree of immunostimulation with no evidence of pathological autoimmunity, we were unable to differentiate between patients with different clinical outcomes based on the parameters measured. Future studies should focus on characterizing more reliable correlations between immune response parameters and clinical outcome that may be used as predictors of vaccine success in immunosuppressed individuals. Cancer Gene Therapy (2009) 16, 598-608; doi:10.1038/cgt.2009.9; published online 6 February 2009

Expression of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 2B, in the bovine corpus luteum

There is evidence that several fibroblast growth factors (FGFs) are involved in growth and development of the corpus luteum (CL), but many FGFs have not been investigated in this tissue, including FGF10. The objective of this study was to determine if FGF10 and its receptor (FGFR2B) are expressed in the CL. Bovine CL were collected from an abattoir and classed as corpus hemorrhagica (stage 1), developing (stage 11), developed (stage 111), and regressed (stage IV) CL. Expression of FGF10 and FGFR2B mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR). Both genes were expressed in bovine CL, and FGF10 expression did not differ between stages of CL development. FGF10 protein was localized to large and small luteal cells by immunohistochemistry. FGFR2B expression was approximately threefold higher in regressed compared to developing and developed CL (P < 0.05). To determine if FGF10 and FGFR2B expression is regulated during functional luteolysis, cattle were injected with PGF2 alpha and CL collected at 0, 0.5, 2, 4, 12, 24, 48, and 64 hr thereafter (n = 5 CL/time point), and mRNA abundance was measured by real-time RT-PCR. FGF10 mRNA expression did not change during functional luteolysis, whereas FGFR2B mRNA abundance decreased significantly at 2, 4, and 12 hr after PGF2a, and returned to pretreatment levels for the period 24-64 hr post-PGF2 alpha. These data suggest a potential role for FGFR2B signaling during structural luteolysis in bovine CL.

Stable expression of noncytopathic Kunjin replicons simulates both ultrastructural and biochemical characteristics observed during replication of Kunjin virus

This report focuses mainly on the characterization of a Vero cell line stably expressing the flavivirus Kunjin (KUN) replicon C20SDrep (C20SDrepVero). We showed by immunofluorescence and cryoimmunoelectron microscopy that unique flavivirus-induced membrane structures, termed convoluted membranes/paracrystalline structures, were induced in the C20SDrepVero cells. These induced cytoplasmic foci were immunolabeled with KUN virus anti-NS3 antibodies and with antibodies to the cellular markers ERGIC53 (for the intermediate compartment) and protein disulfide isomerase (for the rough endoplasmic reticulum). However, in contrast to the large perinuclear inclusions observed by immunofluorescence with anti-double-stranded (ds)RNA antibodies in KUN virus-infected cells, the dsRNA in C20SDrepVero cells was localized to small isolated foci scattered throughout the cytoplasm, which were coincident with small foci dual-labeled with the trans-Golgi specific marker GaIT. importantly persistent expression of the KUN replicons in cells did not produce cytopathic effects, and the morphology of major host organelles (including Golgi, mitochondria, endoplasmic reticulum, and nucleus) was apparently unaffected. The amounts of plus- and minus-sense RNA synthesis in replicon cells were similar to those in KUN virus-infected cells until near the end of the latent period, but subsequently increases of about 10- and fourfold, respectively, occurred in infected cells. Virus-specified protein synthesis in C20SDrepVero cells was also about 10-fold greater than that in infected cells. When several KUN replicon cell lines were compared with respect to membrane induction, the relative efficiencies increased in parallel with increases in viral RNA and protein synthesis, consistent with the increases observed during the virus infectious cycle. Based on these observations, cell lines expressing less-efficient replicons may provide a useful tool to study early events in flavivirus RNA replication, which are difficult to assess in Virus infections. (C) 2001 Academic press.

A common inhibitory binding site for zinc and odorants at the voltage-gated K+ channel of rat olfactory receptor neurons

This study compared the effects of zinc and odorants on the voltage-gated K+ channel of rat olfactory neurons. Zinc reduced current magnitude, depolarized the voltage activation curve and slowed activation kinetics without affecting inactivation or deactivation kinetics. Zinc inhibition was potentiated by the NO compound, S-nitroso-cysteine. The pH- and diethylpyrocarbonate-dependence of zinc inhibition suggested that zinc acted by binding to histidine residues. Cysteine residues were eliminated as contributing to the zinc-binding site. The odorants, acetophenone and amyl acetate, also reduced current magnitude, depolarized the voltage activation curve and selectively slowed activation kinetics. Furthermore, the diethylpyrocarbonate- and pH-dependence of odorant inhibition implied that the odorants also bind to histidine residues. Zinc inhibitory potency was dramatically diminished in the presence of odorants, implying competition for a common binding site. These observations indicate that the odorants and zinc share a common inhibitory binding site on the external surface of the voltage-gated K+ channel.

The VCAM-1 gene that encodes the vascular cell adhesion molecule is a target of the Sry-related high mobility group box gene, Sox18

VCAM-1 (vascular cell adhesion molecule-1) and Sox18 are involved in vascular development. VCAM-1 is an important adhesion molecule that is expressed on endothelial cells and has a critical role in endothelial activation, inflammation, lymphatic pathophysiology, and atherogenesis. The Sry-related high mobility group box factor Sox18 has previously been implicated in endothelial pathologies. Mutations in human and mouse Sox18 leads to hypotrichosis and lymphedema. Furthermore, both Sox18 and VCAM-1 have very similar spatio-temporal patterns of expression, which is suggestive of crosstalk. We use biochemical techniques, cell culture systems, and the ragged opossum (RaOP) mouse model with a naturally occurring mutation in Sox18 to demonstrate that VCAM-1 is an important target of Sox18. Transfection, site-specific mutagenesis, and gel shift analyses demonstrated that Sox18 directly targeted and trans-activated VCAM-1 expression. Importantly, the naturally occurring Sox18 mutant attenuates the expression and activation of VCAM-1 in vitro. Furthermore, in vivo quantitation of VCAM-1 mRNA levels in wild type and RaOP mice demonstrates that RaOP animals show a dramatic and significant reduction in VCAM-1 mRNA expression in lung, skin, and skeletal muscle. Our observation that the VCAM-1 gene is an important target of SOX18 provides the first molecular insights into the vascular abnormalities in the mouse mutant ragged and the human hypotrichosis-lymphedematelangiectasia disorder.

Expanding our therapeutic options: β-blockers for colon cancer?

Supported by U. Porto/Santander Totta (IJUP) (PP-IJUP2011-320)

In vitro assessment of three dimensional dense chitosan-based structures to be as bioabsorbable implants

Chitosan biocompatibility and biodegradability properties make this biopolymer promising for the development of advanced internal fixation devices for orthopedic applications. This work presents a detailed study on the production and characterization of three dimensional (3D) dense, non-porous, chitosan-based structures, with the ability to be processed in different shapes, and also with high strength and stiffness. Such features are crucial for the application of such 3D structures as bioabsorbable implantable devices. The influence of chitosan's molecular weight and the addition of one plasticizer (glycerol) on 3D dense chitosan-based products' biomechanical properties were explored. Several specimens were produced and in vitro studies were performed in order to assess the cytotoxicity of these specimens and their physical behavior throughout the enzymatic degradation experiments. The results point out that glycerol does not impact on cytotoxicity and has a high impact in improving mechanical properties, both elasticity and compressive strength. In addition, human mesenchymal stem/stromal cells (MSC) were used as an ex-vivo model to study cell adhesion and proliferation on these structures, showing promising results with fold increase values in total cell number similar to the ones obtained in standard cell culture flasks. (C) 2014 Elsevier Ltd. All rights reserved.

Estudo da Toxicidade do Chumbo na Levedura Saccharomyces cerevisiae

O chumbo é um importante poluente ambiental. A levedura Saccharomyces cerevisiae constitui um modelo útil para o estudo dos efeitos tóxicos do chumbo. O conhecimento dos mecanismos de defesa e resistência à presença de metais pesados poderá ser útil em tecnologias de proteção ambiental, nomeadamente no desenvolvimento de novas metodologias para a biorremediação de metais pesados. O presente trabalho teve como objetivo avaliar o impacto do Pb na capacidade proliferativa, na integridade membranar e na produção intracelular de espécies reativas de oxigénio (ROS), na estirpe laboratorial da levedura Saccharomyces cerevisiae BY4741 (estirpe selvagem, WT). Foi também estudado o papel das mitocôndrias, como fonte de ROS induzida por Pb, e o envolvimento da H+-ATPase vacuolar (V-ATPase) e de transportadores vacuolares pertencentes à superfamília ABC (de ATP-binding cassette) na defesa contra a toxicidade do Pb. O estudo cinético do impacto de duas concentrações de Pb na viabilidade das leveduras (avaliado através de um ensaio clonogénico), na integridade da membrana celular (determinada com iodeto de propídio) e na produção intracelular de ROS (o anião superóxido foi detetado com dihidroetídio e o peróxido de hidrogénio com 2’,7’- diclorodihidrofluoresceína), revelou uma perda progressiva da capacidade proliferativa (53 e 17% de células viáveis, após a exposição durante 3h a 250 ou 1000 µmol/l de chumbo, respetivamente), coincidente com a acumulação intracelular de anião superóxido e de peróxido de hidrogénio, na ausência de perda da integridade membranar. A importância das mitocôndrias na produção de ROS, induzida por chumbo, foi levada a cabo usando um mutante deficiente respiratório desprovido de ADN mitocondrial (ƿ0). Quando comparado com a respetiva estirpe parental, o mutante ƿ0 apresentou uma maior resistência ao Pb e uma menor produção de ROS induzida por Pb. A exposição das células da estirpe BY4741 a 250 e 1000 µmol/l de chumbo originou a formação de 49 e 58% de células deficientes respiratórias, respetivamente. A função da V-ATPase, na desintoxicação de chumbo, foi avaliada utilizando mutantes com uma estrutura vacuolar normal mas defetivos em subunidades da VATPase (vma1Δ, vma2Δ, vma3Δ e vph1Δ). Comparativamente às células da estirpe WT, todos os mutantes testados, sem V-ATPase funcional, apresentaram uma maior suscetibilidade ao Pb. O papel dos transportadores vacuolares pertencentes à superfamília ABC, na defesa contra a toxicidade induzida por chumbo, foi levada a cabo utilizando mutantes sem os transportadores Ycf1p ou Vmr1p. Os resultados preliminares mostraram que quando comparadas com as células da estirpe WT, as células das estirpes ycf1Δ ou vmr1Δ não apresentavam uma maior perda da viabilidade. A modificação da morfologia vacuolar, em células expostas a chumbo, foi visualizada utilizando a estirpe Vma2p-GFP. O tratamento das células com Pb originou a fusão dos vacúolos de tamanho médio num único vacúolo de grande dimensão. Em conclusão, os estudos desenvolvidos no presente trabalho, utilizando a estirpe laboratorial BY4741, mostraram que a perda da capacidade proliferativa das leveduras, induzida pelo chumbo, pode ser atribuída à acumulação intracelular do anião superóxido e de peróxido de hidrogénio. As mitocôndrias parecem ser uma das principais fontes de ROS induzido por Pb e, simultaneamente, um dos principais alvos da sua toxicidade. Em S. cerevisiae, o vacúolo desempenha um papel importante na desintoxicação do Pb. A modificação da morfologia vacuolar após exposição ao chumbo poderá ser a consequência da acumulação de Pb no vacúolo. Enquanto os transportadores da superfamília ABC parecem não estar envolvidos na sequestração vacuolar de Pb, é necessária a presença, num estado funcional, da V-ATPase para que ocorra a compartimentação do Pb. Muito provavelmente, a compartimentação do Pb no vacúolo previne a sua acumulação no citosol e o desencadear dos respetivos efeitos tóxicos.

Multiphasic, multistructured and hierarchical strategies for cartilage regeneration

Cartilage tissue is a complex nonlinear, viscoelastic, anisotropic, and multiphasic material with a very low coefficient of friction, which allows to withstand millions of cycles of joint loading over decades of wear. Upon damage, cartilage tissue has a low self-reparative capacity due to the lack of neural connections, vascularization, and a latent pool of stem/chondroprogenitor cells. Therefore, the healing of articular cartilage defects remains a significant clinical challenge, affecting millions of people worldwide. A plethora of biomaterials have been proposed to fabricate devices for cartilage regeneration, assuming a wide range of forms and structures, such as sponges, hydrogels, capsules, fibers, and microparticles. In common, the fabricated devices were designed taking in consideration that to fully achieve the regeneration of functional cartilage it is mandatory a well-orchestrated interplay of biomechanical properties, unique hierarchical structures, extracellular matrix (ECM), and bioactive factors. In fact, the main challenge in cartilage tissue engineering is to design an engineered device able to mimic the highly organized zonal architecture of articular cartilage, specifically its spatiomechanical properties and ECM composition, while inducing chondrogenesis, either by the proliferation of chondrocytes or by stimulating the chondrogenic differentiation  of stem/chondro-progenitor cells. In this chapter we present the recent advances in the development of innovative and complex biomaterials that fulfill the required structural key elements for cartilage regeneration. In particular, multiphasic, multiscale, multilayered, and hierarchical strategies composed by single or multiple biomaterials combined in a welldefined structure will be addressed. Those strategies include biomimetic scaffolds mimicking the structure of articular cartilage or engineered scaffolds as models of research to fully understand the biological mechanisms that influence the regeneration of cartilage tissue.

Impact of mutated KRAS signalling on autophagy regulation in colorectal carcinoma

Programa Doutoral em Biologia Molecular e Ambiental

Current approaches and future perspectives on strategies for the development of personalized tissue engineering therapies

Personalized tissue engineering and regenerative medicine (TERM) therapies propose patient-oriented effective solutions, considering individual needs. Cell-based therapies, for example, may benefit from cell sources that enable easier autologous set-ups or from recent developments on IPS cells technologies towards effective personalized therapeutics. Furthermore, the customization of scaffold materials to perfectly fit a patientâ s tissue defect through rapid prototyping technologies, also known as 3D printing, is now a reality. Nevertheless, the timing to expand cells or to obtain functional in vitrotissue substitutes prior to implantation prevents advancements towards routine use upon patient´s needs. Thus, personalized therapies also anticipate the importance of creating off-the-shelf solutions to enable immediately available tissue engineered products. This paper reviews the main recent developments and future challenges to enable personalized TERM approaches and to bring these technologies closer to clinical applications.

Functional sphere profiling reveals the complexity of neuroblastoma tumor-initiating cell model.

Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically "profile" the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB.

Genetic dissection of c-Myc function during mouse organogenesis

Résumé Le gène c-myc est un des oncogènes les plus fréquemment mutés dans les tumeurs humaines. Même si plus de 70 % des cancers humains montrent une dérégulation de c-Myc, les connaissances sur son rôle physiologique pendant le développement, et dans la souris adulte restent très peu connus. Récemment, notre laboratoire a pu montrer que c-Myc contrôle l'équilibre entre le renouvellement et la différenciation des cellules souches hématopoïetiques (CSH) dans la souris adulte. Ceci est probablement dû à lacapacité de c-Myc de contrôler l'entrée et la sortie des CSH de leur niche de la moelle osseuse, en régulant plusieurs molécules d'adhésion, parmi lesquelles la cadhérine-N (Wilson et al., 2004; Wilson and Trumpp, 2006). Des études utilisant un mutant d'inactivation ont demontré que la protéine c-Myc est essentielle pour le développement au delà du jour embryonnaire E9.5. Les embryons c-Myc déficients sont plus petits que la normale et possèdent de nombreux défauts; en particulier ils ne peuvent établir un système hématopoietique embryonnaire primitif (Trumpp et al., 2001). Nous avons récemment découvert que le développement du placenta dépend de la présence de cMyc. Ceci permet de proposer que certains, sinon tous, les défauts embryonnaires puorraient dériver indirectement d'un défaut nutritionnel causé par la défaillance du placenta. Afin de répondre à cette question de manière génétique, nous avons utilisé l'allele conditionel c-mycflox (Trumpp et al., 2001) en combinaison avec l'allele Sox2-Cre (Hayashi et al., 2002). Celui-ci détermine l'expression de la récombinase Cre spécifiquement dans les cellules de l'épiblaste à partir de E6.5, tandis qu'il n'y a pas, ou seulement très peu, d'activité de la récombinase Cre dans les tissus extraembryonnaires.Alnsi, cette stratégie nous permet de générer des embryons sans c-Myc qui se développent en présence d'un compartment extraembryonnaire ou c-Myc est exprimé normalement (Sox2Cre;c-mycflox2) Ces embryons, Sox2Cre;c-mycflox2 se développent et grandissent normalement tout en formant un système vasculaire normal, mais meurent à E11.5 à cause d'un sévère manque de cellules hématopoïetiques. De façon très intéressante, la seule population qui semble être présente en nombre à peu près normal dans ces embryons est celle des précurseurs et des cellules souches. Les cellules qui forment cette population prolifèrent normalement mais ne peuvent pas former des colonies in vitro, ce qui montre que ces cellules ont perdu leur activité de cellules souches. Cependant, lorsque nous avons analysé ces cellules plus en détail en éxaminant l'expression des molécules d'intégrine nous avons découvert que l'integrine ß est sur-éxprimée à la surface des cellules c-Myc déficientes. Ceci pourrait indiquer un mécanisme par lequel c-Myc régule des molécules d'adhésion sur les cellules du sang. En conséquence, en absence de c-Myc, l'adhésion et la migration des cellules du sang de l'AGM (Aorte-Gonade-Mésonéphros) vers le foie de l'embryon, à travers le système vasculaire, est compromise. En outre, nous avons pu montrer que les hépatocytes du foie, qui constitue le site principal de formation des cellules hématopoïetiques pendant le développement, est sévèrement atteint dans des Sox2Cre;c-mycflox2 embryons. Ceci n'est pas du à un défaut propre aux cellules hépatiques qui ont perdu c-Myc, mais résulte plutôt de l'absence de cellules hématopoietïques qui normalement colonisent le foie à ce stade du développement. Ces résultats représentent la première preuve directe que le développement des hépatoblastes est dépendant de signaux provenant des cellules du sang. Summary The myc gene is one of the most frequently mutated oncogenes in human tumors. It is found to be mis-regulated in over 70% of all human cancers. However, our knowledge about its physiological role in mammalian development and adulthood remains limited. Recent work in our laboratory showed that c-Myc controls the balance between hematopoietic stem cell (HSC) self-renewal and differentiation in the adult mouse. This is likely due to the capacity of c-Myc to control entry and exit of HSCs from the bone marrow niche by regulating a number of cell adhesion molecules including N-cadherin (Wilson et al., 2004; Wilson and Trumpp 2006). During development knockout studies showed that c-Myc is required for embryonic development beyond embryonic day (E) 9.5. c-Myc deficient embryos are severely reduced in size and show multiple defects including the failure to establish a primitive hematopoietic system (Trumpp et al., 2001). Importantly, we recentry uncovered that placental development also seems to depend on normal c-Myc function, raising the possibility that some if not all of the embryonic defects observed could be mediated indirectly by a nutrition defect caused by placental failure. To address this possibility genetically, we took advantage of the conditional c-mycflox allele (Trumpp et al., 2001) in combination with the Sox2-Cre allele (Hayashi et al., 2002), in which Cre expression is specifically targeted to all epiblast cells by E6.5, while there is little or no Cre activity inextra-embryonic lineages. Thus, this strategy allows the generation of c-Myc deficient embryos, which develop within a normal c-Myc expressing extra-embryonic compartment (Sox2Cre;c-mycflox2) Such Sox2Cre;c-mycflox2 embryos develop and grow appropriately and form a normal vascular system but die at E11.5 due to a severe lack of blood cells. Interestingly, the only hematopoietic population that seems to be present in almost normal numbers in the embryo is the stem/progenitor cell population. Cells within this populatíon proliferate normal but can not give rise to hematopoietic colonies in vitro showing that functional hematopoietic stem cell (HSC) activity is lost. However, when we analyzed these phenotypic HSCs in more detail and examined integrin expression in mutant stem/progenitor cells, we observed that ß1-integrin is upregulated. This may point to a potential mechanism whereby c-Myc regulates adhesíon molecules on hematopoietic cells and thereby disturbs adhesion and migration from the AGM (aorta-gonads-mesonephros) through the vascular system to the liver. Furthermore, we uncovered that the fetal liver, the main site of hematopoietic expansion at that stage, is severely affected in Sox2Cre;c-mycflox2 embryos and that this is not due to a cell intrinsic defect of c-Myc deficient hepatocytes but rather due to the lack of hematopoietic cells that normally colonize the fetal liver at that stage of development. This provides first direct evidence that hepatoblast development depends on signals derived from blood cells.