Potential Effect of Pseudevernia furfuracea (L.) Zopf Extract and Metabolite Physodic Acid on Tumour Microenvironment Modulation in MCF-10A Cells.

Lichens comprise a number of unique secondary metabolites with remarkable biological activities and have become an interesting research topic for cancer therapy. However, only a few of these metabolites have been assessed for their effectiveness against various in vitro models. Therefore, the aim of the present study was to assess the effect of extract Pseudevernia furfuracea (L.) Zopf (PSE) and its metabolite physodic acid (Phy) on tumour microenvironment (TME) modulation, focusing on epithelial-mesenchymal transition (EMT), cancer-associated fibroblasts (CAFs) transformation and angiogenesis. Here, we demonstrate, by using flow cytometry, Western blot and immunofluorescence microscopy, that tested compounds inhibited the EMT process in MCF-10A breast cells through decreasing the level of different mesenchymal markers in a time- and dose-dependent manner. By the same mechanisms, PSE and Phy suppressed the function of Transforming growth factor beta (TGF-ß)-stimulated fibroblasts. Moreover, PSE and Phy resulted in a decreasing level of the TGF-ß canonical pathway Smad2/3, which is essential for tumour growth. Furthermore, PSE and Phy inhibited angiogenesis ex ovo in a quail embryo chorioallantoic model, which indicates their potential anti-angiogenic activity. These results also provided the first evidence of the modulation of TME by these substances.

Relevance of angiogenesis in autoimmune testis inflammation.

Experimental autoimmune orchitis (EAO) is a useful model to study organ-specific autoimmunity and chronic testicular inflammation. This model reflects testicular pathological changes reported in immunological infertility in men. Progression of EAO in rodents is associated with a significantly increased percentage of testicular endothelial cells and interstitial testicular blood vessels, indicating an ongoing angiogenic process. Vascular endothelial growth factor A (VEGFA), the main regulator of physiological and pathological angiogenesis, can stimulate endothelial cell proliferation, chemotaxis and vascular permeability. The aim of this study was to explore the role of VEGFA in the pathogenesis of testicular inflammation. Our results found VEGFA expression in Leydig cells, endothelial cells and macrophages in testis of rats with autoimmune orchitis. VEGFA level was significantly higher in testicular fluid and serum of rats at the end of the immunization period, preceding testicular damage. VEGF receptor (VEGFR) 1 is expressed mainly in testicular endothelial cells, whereas VEGFR2 was detected in germ cells and vascular smooth muscle cells. Both receptors were expressed in testicular interstitial cells. VEGFR2 increased after the immunization period in the testicular interstitium and VEGFR1 was downregulated in EAO testis. In-vivo-specific VEGFA inhibition by Bevacizumab prevented the increase in blood vessel number and reduced EAO incidence and severity. Our results unveil relevance of VEGFA-VEGFR axis during orchitis development, suggesting that VEGFA might be an early marker of testicular inflammation and Bevacizumab a therapeutic tool for treatment of testicular inflammation associated with subfertility and infertility.

PuraMatrix allows differentiation of a broad repertoire of neural and mesenchymal phenotypes from trunk neural crest.

The neural crest (NC) is a transitory embryonic structure of vertebrates that gives rise to an astonishing variety of derivatives, encompassing both neural and mesenchymal cell types. Neural crest cells (NCCs) are an excellent model to study how environmental factors modulate features such as cell multipotentiality and differentiation. Tests with multifunctional substrates that allow NCCs to express their full potential, while promoting cell subcloning, are needed to advance knowledge about NCC self-renewal and to foster future biotechnological approaches. Here we show that a self-assembled peptide named PuraMatrixTM is an excellent substrate that allows the differentiation of NCCs based on the identification of seven different cell types. Depending on the PuraMatrixTM concentration employed, different frequencies and quantities of a given cell type were obtained. It is noteworthy that an enormous quantity and diversity of mesenchymal phenotypes, such as chondrocytes, could be observed. The quantity of adipocytes and osteocytes also increased with the use of mesenchymal differentiation factors (MDF), but PuraMatrixTM alone can support the appearance of these mesenchymal cell types. PuraMatrixTM will promote advances in studies related to multipotentiality, self-renewal and control of NCC differentiation, since it is an extremely simple and versatile material which can be employed for both in vivo and in vitro experiments.

Low-dose photodynamic therapy promotes angiogenic potential and increases immunogenicity of human mesenchymal stromal cells.

Photodynamic therapy (PDT) is a non-invasive FDA and EMA-approved anticancer treatment modality. Initially developed for elimination of malignant cells, PDT affects all cells in the tumor bed including stromal cells. Stroma represents not only an important component of tumor microenvironment, but has a significant impact on tumor susceptibility to PDT and other anticancer therapies. However, the effects of PDT on stromal cells are poorly investigated. During PDT the tumor stroma can receive low-dose irradiation as a result of chosen regimen or limited depth of light penetration. Here, we characterized response of human mesenchymal stromal cells (MSCs) to low-dose PDT. In an in vitro model we demonstrated that low-dose PDT resulted in activation of Erk1/2 and inhibition of GSK-3 signaling in MSCs. PDT-mediated induction of intracellular reactive oxygen species (ROS) resulted in reorganization of MSC cytoskeleton and decreased cell motility. More importantly, low-dose PDT dramatically upregulated secretion of various proangiogenic factors (VEGF-A, IL-8, PAI-1, MMP-9, etc.) by MSCs and improved MSC ability to promote angiogenesis suggesting an increase in the pro-tumorigenic potential of MSCs. In contrast, co-cultivation of PDT-treated MSCs with lymphocytes resulted in significant decrease of MSC viability and potential increase in MSC immunogenicity, which may lead to increased anti-tumor immunity. Low-dose PDT in MSCs significantly inhibited secretion of CCL2 (MCP-1) potentially limiting infiltration of pro-tumorigenic macrophages. Altogether, our findings demonstrate that low-dose PDT significantly modifies functional properties of MSCs improving their pro-tumorigenic potential while simultaneously increasing potential immune stimulation suggesting possible mechanisms of stromal cell contribution to PDT efficacy.

Nicole Le Douarin and the use of quail-chick chimeras to study the developmental fate of neural crest and hematopoietic cells.

The quail-chick chimera marking system, devised in 1969, gave a new impetus to the analysis of cell migrations and interactions in the developing nervous, immune and hematopoietic systems. The method is based on the observation that the constitutive heterochromatin in all embryonic and adult cells of the quail is condensed in one large mass in the centre of the nucleus and is associated with the nucleolus, making this organelle strongly stained with the Feulgen-Rossenbeck reaction. The association of cells or rudiments from two avian species, advocated as a means to identify cells that migrate during embryogenesis, was rapidly recognized in this context as a useful tool for the study of many developmental biology problems. This article summarizes the fundamental contribution of Nicole Le Douarin to the discovery and the application of this technique over the last 40 years.

Altered VEGF Signaling Leads to Defects in Heart Tube Elongation and Omphalomesenteric Vein Fusion in Quail Embryos.

Formation of the endocardial and myocardial heart tubes involves precise cardiac progenitor sorting and tissue displacements from the primary heart field to the embryonic midline-a process that is dependent on proper formation of conjoining great vessels, including the omphalomesenteric veins (OVs) and dorsal aortae. Using a combination of vascular endothelial growth factor (VEGF) over- and under-activation, fluorescence labeling of cardiac progenitors (endocardial and myocardial), and time-lapse imaging, we show that altering VEGF signaling results in previously unreported myocardial, in addition to vascular and endocardial phenotypes. Resultant data show: (1) exogenous VEGF leads to truncated endocardial and myocardial heart tubes and grossly dilated OVs; (2) decreased levels of VEGF receptor 2 tyrosine kinase signaling result in a severe abrogation of the endocardial tube, dorsal aortae, and OVs. Surprisingly, only slightly altered myocardial tube fusion and morphogenesis is observed. We conclude that VEGF has direct effects on the VEGF receptor 2-bearing endocardial and endothelial precursors, and that altered vascular morphology of the OVs also indirectly results in altered myocardial tube formation. Anat Rec, 302:175-185, 2019. © 2018 Wiley Periodicals, Inc.

Oxidative and mutagenic effects of low intensity GSM 1800 MHz microwave radiation.

AIM: Despite a significant number of epidemiological studies on potential carcinogenicity of microwave radiation (MWR) from wireless devices and a bulk of experimental studies on oxidative and mutagenic effects of low intensity MWR, the discussion on potential carcinogenicity of low intensity MWR is going on. This study aims to assess oxidative and mutagenic effects of low intensity MWR from a typical commercial model of a modern smartphone. MATERIALS AND METHODS: The model of developing quail embryos has been used for the assessment of oxidative and mutagenic effects of Global System for Mobile communication (GSM) 1800 MHz MWR from a commercial model of smartphone. The embryos were exposed in ovo to 0.32 µW/cm2, discontinuously - 48 s - On, 12 s - Off, during 5 days before and 14 days through the incubation period. RESULTS: The exposure of quail embryos before and during the incubation period to low intensity GSM 1800 MHz has resulted in expressive statistically significant oxidative effects in embryonic cells, including a 2-fold increase in superoxide generation rate and 85% increase in nitrogen oxide generation rate, damages of DNA integrity and oxidative damages of DNA (up to twice increased levels of 8-oxo-dG in cells of 1-day old chicks from the exposed embryos). Finally, the exposure resulted in a significant, almost twice, increase of embryo mortality. CONCLUSION: The exposure of model biological system to low intensity GSM 1800 MHz MWR resulted in significant oxidative and mutagenic effects in exposed cells, and thus should be recognized as a significant risk factor for living cells.

General principles of spinal motor circuit development: early contributions from research on avian embryos.

Birds and mammals, both being amniotes, share many common aspects of development. Thus our understanding of how limb-innervating mammalian spinal motor circuits develop was greatly influenced by the use of the avian embryo (chick/quail) to bring about experimental perturbations to identify basic underlying mechanisms. These included embryonic surgery, the application of drugs to influence activity or molecular interactions, and the ability to observe motor behavior and make physiological recordings in intact developing embryos. This article will review some of these contributions, highlighting several areas including the acquisition of motoneuron subtype identity and target selection, as well as the role of spontaneous rhythmic activity in circuit development.

Avian coronary endothelium is a mosaic of sinus venosus- and ventricle-derived endothelial cells in a region-specific manner.

The origin of coronary endothelial cells (ECs) has been investigated in avian species, and the results showed that the coronary ECs originate from the proepicardial organ (PEO) and developing epicardium. Genetic approaches in mouse models showed that the major source of coronary ECs is the sinus venosus endothelium or ventricular endocardium. To clarify and reconcile the differences between avian and mouse species, we examined the source of coronary ECs in avian embryonic hearts. Using an enhanced green fluorescent protein-Tol2 system and fluorescent dye labeling, four types of quail-chick chimeras were made and quail-specific endothelial marker (QH1) immunohistochemistry was performed. The developing PEO consisted of at least two cellular populations in origin, one was sinus venosus endothelium-derived inner cells and the other was surface mesothelium-derived cells. The majority of ECs in the coronary stems, ventricular free wall, and dorsal ventricular septum originated from the sinus venosus endothelium. The ventricular endocardium contributed mainly to the septal artery and a few cells to the coronary stems. Surface mesothelial cells of the PEO differentiated mainly into a smooth muscle phenotype, but a few differentiated into ECs. In avian species, the coronary endothelium had a heterogeneous origin in a region-specific manner, and the sources of ECs were basically the same as those observed in mice.

Immunohistochemical localization of estrogen receptor in the embryonic gonad of male quail embryo during gonadal differentiation / Localización inmunohistoquímica del receptor de estrógeno en la gónada embrionaria en el embrión de codorniz macho durante la diferenciación gonadal

In birds, male embryo the gonads develop bilateral testes, in which both left and right sides produce functional spermatozoa, whereas female embryo, only the left gonad develops into a functional ovary. Estrogen plays a key role in avian sex determination in both sexes by binding to the estrogen receptor (ER). Surprisingly, chicken estrogen receptor (cER) mRNA is expressed in both sexes; moreover; its expression is only expressed in the left male gonad. The present study aimed to localize ER protein in the left gonad of male quail embryo using immunohistochemistry. The 8-day-old male quail embryos whose embryonic sex distinguished by gonadal morphology were studied. Histology of the left male gonad displayed thin cortex containing 1 to 2 layers of the germinal epithelium, while testicular cords were observed in the medulla. ER-immunoreactive cells were only found in the germinal epithelium but not in the medulla. Localization of ER was detected in the nucleus and cytoplasm of the germinal epithelial cells. The number of ER-immunoreactive cells counted in upper, lateral, and lower regions of the germinal epithelium was 18.20±1.892, 17.60±1.887, and 16.20±1.290, respectively. This study shows the first evidence for expression of ER protein in the left male gonad of the avian embryo, indicating that ER plays a role in avian gonadal sex differentiation.

En las aves, la gónada embrionaria en los machos se desarrolla bilateralmente, ambos testículos producen espermatozoides funcionales, mientras que en el embrión hembra, sólo la gónada izquierda se convierte en un ovario funcional. El estrógeno juega un papel clave en la determinación del sexo aviar, en ambos sexos, mediante la unión al receptor de estrógeno (RE). Fuertemente los receptores de estrógenos de pollo (cRE) el ARNm se expresan en ambos sexos; además, su expresión sólo se produce en la gónada izquierda del macho. El objetivo fue localizar proteínas del RE en la gónada izquierda de embriones de codorniz macho mediante inmunohistoquímica. Se estudiaron embriones de codorniz machos a los 8 días de edad, cuyo sexo embrionario se distinguió por la morfología de las gónadas. La histología de la gónada izquierda estuvo representada por la corteza delgada que contiene de 1 a 2 capas del epitelio germinal, mientras que se observaron cordones testiculares en la médula. El RE se encontró en células inmunorreactivas del epitelio germinal, pero no en la médula. Se detectó la localización de RE en el núcleo y el citoplasma de las células epiteliales germinales. El número de células RE-inmunorreactivas en las regiones superior, lateral e inferior del epitelio germinal fue de 18,20±1,892, 17,60±1,887 y 16,20±1,290, respectivamente. Este estudio muestra la primera evidencia de expresión de la proteína de RE en la gónada izquierda del embrión aviar macho, lo que indica que el RE desempeña un papel en la diferenciación sexual de la gónada aviar.

Sema3A maintains corneal avascularity during development by inhibiting Vegf induced angioblast migration.

Corneal avascularity is important for optical clarity and normal vision. However, the molecular mechanisms that prevent angioblast migration and vascularization of the developing cornea are not clear. Previously we showed that periocular angioblasts and forming ocular blood vessels avoid the presumptive cornea despite dynamic ingression of neural crest cells. In the current study, we investigate the role of Semaphorin3A (Sema3A), a cell guidance chemorepellent, on angioblast migration and corneal avascularity during development. We show that Sema3A, Vegf, and Nrp1 are expressed in the anterior eye during cornea development. Sema3A mRNA transcripts are expressed at significantly higher levels than Vegf in the lens that is positioned adjacent to the presumptive cornea. Blockade of Sema3A signaling via lens removal or injection of a synthetic Sema3A inhibitor causes ectopic migration of angioblasts into the cornea and results in its subsequent vascularization. In addition, using bead implantation, we demonstrate that exogenous Sema3A protein inhibits Vegf-induced vascularization of the cornea. In agreement with these findings, loss of Sema/Nrp1 signaling in Nrp1(Sema-) mutant mice results in ectopic angioblasts and vascularization of the embryonic mouse corneas. Altogether, our results reveal Sema3A signaling as an important cue during the establishment of corneal avascularity in both chick and mouse embryos. Our study introduces cornea development as a new model for studying the mechanisms involved in vascular patterning during embryogenesis and it also provides new insights into therapeutic potential for Sema3A in neovascular diseases.

Environmental factors unveil dormant developmental capacities in multipotent progenitors of the trunk neural crest.

The neural crest (NC), an ectoderm-derived structure of the vertebrate embryo, gives rise to the melanocytes, most of the peripheral nervous system and the craniofacial mesenchymal tissues (i.e., connective, bone, cartilage and fat cells). In the trunk of Amniotes, no mesenchymal tissues are derived from the NC. In certain in vitro conditions however, avian and murine trunk NC cells (TNCCs) displayed a limited mesenchymal differentiation capacity. Whether this capacity originates from committed precursors or from multipotent TNCCs was unknown. Here, we further investigated the potential of TNCCs to develop into mesenchymal cell types in vitro. We found that, in fact, quail TNCCs exhibit a high ability to differentiate into myofibroblasts, chondrocytes, lipid-laden adipocytes and mineralizing osteoblasts. In single cell cultures, both mesenchymal and neural cell types coexisted in TNCC clonal progeny: 78% of single cells yielded osteoblasts together with glial cells and neurons; moreover, TNCCs generated heterogenous clones with adipocytes, myofibroblasts, melanocytes and/or glial cells. Therefore, alike cephalic NCCs, early migratory TNCCs comprised multipotent progenitors able to generate both mesenchymal and melanocytic/neural derivatives, suggesting a continuum in NC developmental potentials along the neural axis. The skeletogenic capacity of the TNC, which was present in the exoskeletal armor of the extinct basal forms of Vertebrates and which persisted in the distal fin rays of extant teleost fish, thus did not totally disappear during vertebrate evolution. Mesenchymal potentials of the TNC, although not fulfilled during development, are still present in a dormant state in Amniotes and can be disclosed in in vitro culture. Whether these potentials are not expressed in vivo due to the presence of inhibitory cues or to the lack of permissive factors in the trunk environment remains to be understood.

The negative influence of high-glucose ambience on neurogenesis in developing quail embryos.

Gestational diabetes is defined as glucose intolerance during pregnancy and it is presented as high blood glucose levels during the onset pregnancy. This condition has an adverse impact on fetal development but the mechanism involved is still not fully understood. In this study, we investigated the effects of high glucose on the developing quail embryo, especially its impact on the development of the nervous system. We established that high glucose altered the central nervous system mophologically, such that neural tube defects (NTDs) developed. In addition, we found that high glucose impaired nerve differentiation at dorsal root ganglia and in the developing limb buds, as revealed by neurofilament (NF) immunofluorescent staining. The dorsal root ganglia are normally derived from neural crest cells (NCCs), so we examine the delamination of NCCs from dorsal side of the neural tube. We established that high glucose was detrimental to the NCCs, in vivo and in vitro. High glucose also negatively affected neural differentiation by reducing the number and length of neurites emanating from neurons in culture. We established that high glucose exposure caused an increase in reactive oxidative species (ROS) generation by primary cultured neurons. We hypothesized that excess ROS was the factor responsible for impairing neuron development and differentiation. We provided evidence for our hypothesis by showing that the addition of vitamin C (a powerful antioxidant) could rescue the damaging effects of high glucose on cultured neurons.

Retinoic acid upregulates ret and induces chain migration and population expansion in vagal neural crest cells to colonise the embryonic gut.

Vagal neural crest cells (VNCCs) arise in the hindbrain, and at (avian) embryonic day (E) 1.5 commence migration through paraxial tissues to reach the foregut as chains of cells 1-2 days later. They then colonise the rest of the gut in a rostrocaudal wave. The chains of migrating cells later resolve into the ganglia of the enteric nervous system. In organ culture, E4.5 VNCCs resident in the gut (termed enteric or ENCC) which have previously encountered vagal paraxial tissues, rapidly colonised aneural gut tissue in large numbers as chains of cells. Within the same timeframe, E1.5 VNCCs not previously exposed to paraxial tissues provided very few cells that entered the gut mesenchyme, and these never formed chains, despite their ability to migrate in paraxial tissue and in conventional cell culture. Exposing VNCCs in vitro to paraxial tissue normally encountered en route to the foregut conferred enteric migratory ability. VNCC after passage through paraxial tissue developed elements of retinoic acid signalling such as Retinoic Acid Binding Protein 1 expression. The paraxial tissue's ability to promote gut colonisation was reproduced by the addition of retinoic acid, or the synthetic retinoid Am80, to VNCCs (but not to trunk NCCs) in organ culture. The retinoic acid receptor antagonist CD 2665 strongly reduced enteric colonisation by E1.5 VNCC and E4.5 ENCCs, at a concentration suggesting RARα signalling. By FACS analysis, retinoic acid application to vagal neural tube and NCCs in vitro upregulated Ret; a Glial-derived-neurotrophic-factor receptor expressed by ENCCs which is necessary for normal enteric colonisation. This shows that early VNCC, although migratory, are incapable of migrating in appropriate chains in gut mesenchyme, but can be primed for this by retinoic acid. This is the first instance of the characteristic form of NCC migration, chain migration, being attributed to the application of a morphogen.

Wnt signal specifies the intrathalamic limit and its organizer properties by regulating Shh induction in the alar plate.

The structural complexity of the brain depends on precise molecular and cellular regulatory mechanisms orchestrated by regional morphogenetic organizers. The thalamic organizer is the zona limitans intrathalamica (ZLI), a transverse linear neuroepithelial domain in the alar plate of the diencephalon. Because of its production of Sonic hedgehog, ZLI acts as a morphogenetic signaling center. Shh is expressed early on in the prosencephalic basal plate and is then gradually activated dorsally within the ZLI. The anteroposterior positioning and the mechanism inducing Shh expression in ZLI cells are still partly unknown, being a subject of controversial interpretations. For instance, separate experimental results have suggested that juxtaposition of prechordal (rostral) and epichordal (caudal) neuroepithelium, anteroposterior encroachment of alar lunatic fringe (L-fng) expression, and/or basal Shh signaling is required for ZLI specification. Here we investigated a key role of Wnt signaling in the molecular regulation of ZLI positioning and Shh expression, using experimental embryology in ovo in the chick. Early Wnt expression in the ZLI regulates Gli3 and L-fng to generate a permissive territory in which Shh is progressively induced by planar signals of the basal plate.

Expression of pro- and anti-angiogenic factors during the formation of the periocular vasculature and development of the avian cornea.

BACKGROUND: During embryonic development, endothelial precursor cells (angioblasts) migrate relatively long distances to form the primary vascular plexus. The migratory behavior of angioblasts and localization of the primitive blood vessels is tightly regulated by pro-angiogenic and anti-angiogenic factors encountered in the embryonic environment. Despite the importance of corneal avascularity to proper vision, it is not known when avascularity is established in the developing cornea and how pro- and anti-angiogenic factors regulate this process. RESULTS AND DISCUSSION: Using Tg(tie1:H2B:eYFP) transgenic quail embryos to visualize fluorescently labeled angioblasts, we show that the presumptive cornea remains avascular despite the invasion of cells from the periocular region where migratory angioblasts reside and form the primary vasculature. Semiquantitative reverse transcriptase polymerase chain reaction analysis and spatiotemporal examination of gene expression revealed that pro- and anti-angiogenic factors were expressed in patterns indicating their potential roles in angioblast guidance. CONCLUSIONS: Our findings show for the first time that chick corneal avascularity is established and maintained during development as the periocular vasculature forms. We also identify potential candidate pro- and anti-angiogenic factors that may play crucial roles during vascular patterning in the anterior eye.

Dorsal aorta formation: separate origins, lateral-to-medial migration, and remodeling.

Blood vessel formation is a highly dynamic tissue-remodeling event that can be observed from early development in vertebrate embryos. Dorsal aortae, the first functional intra-embryonic blood vessels, arise as two separate bilateral vessels in the trunk and undergo lateral-to-medial translocation, eventually fusing into a single large vessel at the midline. After this dramatic remodeling, the dorsal aorta generates hematopoietic stem cells. The dorsal aorta is a good model to use to increase our understanding of the mechanisms controlling the establishment and remodeling of larger blood vessels in vivo. Because of the easy accessibility to the developing circulatory system, quail and chick embryos have been widely used for studies on blood vessel formation. In particular, the mapping of endothelial cell origins has been performed using quail-chick chimera analysis, revealing endothelial, vascular smooth muscle, and hematopoietic cell progenitors of the dorsal aorta. The avian embryo model also allows conditional gene activation/inactivation and direct observation of cell behaviors during dorsal aorta formation. This allows a better understanding of the molecular mechanisms underlying specific morphogenetic events during dynamic dorsal aorta formation from a cell behavior perspective.

Distinct roles for N-Cadherin linked c-Src and fyn kinases in lens development.

BACKGROUND: Src family tyrosine kinases (SFKs) are often coincidently expressed but few studies have dissected their individual functions in the same cell during development. Using the classical embryonic lens as our model, we investigated SFK signaling in the regulation of both differentiation initiation and morphogenesis, and the distinct functions of c-Src and Fyn in these processes. RESULTS: Blocking SFK activity with the highly specific inhibitor PP1 induced initiation of the lens differentiation program but blocked lens fiber cell elongation and organization into mini lens-like structures called lentoids. These dichotomous roles for SFK signaling were discovered to reflect distinct functions of c-Src and Fyn and their differentiation-state-specific recruitment to and action at N-cadherin junctions. c-Src was highly associated with the nascent N-cadherin junctions of undifferentiated lens epithelial cells. Its siRNA knockdown promoted N-cadherin junctional maturation, blocked proliferation, and induced lens cell differentiation. In contrast, Fyn was recruited to mature N-cadherin junctions of differentiating lens cells and siRNA knockdown suppressed differentiation-specific gene expression and blocked morphogenesis. CONCLUSIONS: Through inhibition of N-cadherin junction maturation, c-Src promotes lens epithelial cell proliferation and the maintenance of the lens epithelial cell undifferentiated state, while Fyn, signaling downstream of mature N-cadherin junctions, promotes lens fiber cell morphogenesis.

Flavonoid hesperidin protects neural crest cells from death caused by aflatoxin B(1).

The neural crest (NC) corresponds to a collection of multipotent and oligopotent progenitors endowed with both neural and mesenchymal potentials. The derivatives of the NC at trunk level include neurons and glial cells of the peripheral nervous system. Despite the well-known influence of aflatoxins on the development of cancer, the issue of whether they also influence NC cells has not been yet addressed. In the present work, we have investigated the effects of aflatoxin B(1) on quail NC cells and the concomitant effects of the flavonoid hesperidin associated with this mycotoxin. We show for the first time that aflatoxin B(1) decreases the viability and the total number of glial and neuronal cells/field, although their proportions in relation to the total number of cells were not altered. Therefore, aflatoxin has no effect on NC differentiation. However, this compound was able to reduce NC proliferation and NC survival. Furthermore, the co-administration of hesperidin, a well-known polyphenolic protector of cell death, partially prevented the effect of aflatoxin B(1) . Taken together, our results demonstrate that aflatoxin B(1) is toxic to NC cells, an effect partially prevented by the flavonoid hesperidin. This study may contribute to the understanding of the effects of these compounds during early embryonic development and offer potentially more assertive diets and treatments for pregnant animals.

Contribution of cells derived from the area pellucida to extraembryonic mesodermal cell lineages in heterospecific quail chick blastodermal chimeras.

The current study has two main objectives: first, to determine if cells derived from the area pellucida are able to populate extraembryonic membranes, and second, to determine if donor cells have the potential to differentiate to endothelial (EC) and hematopoietic cells (HC) in the yolk sac and allantois, the two extraembryonic membranes functioning as hematopoietic organs in the avian embryo. To this end, quail chick chimeras were constructed by transferring dissociated cells from the areae pellucidae of the stage X-XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in the allantois, yolk sac, amnion, and chorion of resulting putative chimeras was examined using quail cell-specific antibody against a perinuclear antigen (QCPN) after 6 days of incubation. The presence of EC, HC, and smooth muscle cells among the QCPN(+) donor cells was examined using QH-1, a quail-specific marker identifying HC and EC and an anti-α-smooth muscle actin antibody. Evidence gathered in the present study demonstrates that quail cells derived from the areae pellucidae are able to populate all of the extraembryonic membranes of resulting heterospecific quail chick chimeras and, most importantly, give rise to HC, EC, and smooth muscle cells, all of the three main mesodermal lineages derived from the posterior mesoderm both in the yolk sac and allantois.