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To determine the function of VEGF-A in nervous system development, we have utilized the Nestin promoter-driven Cre recombinase transgene, in conjunction with a conditional and hypomorphic VEGF-A allele, to lower VEGF-A activity in neural progenitor cells. Mice with intermediate levels of VEGF-A activity showed decreased blood vessel branching and density in the cortex and retina, resulting in a thinner retina and aberrant structural organization of the cortex. Severe reductions in VEGF-A led to decreases in vascularity and subsequent hypoxia, resulting in the specific degeneration of the cerebral cortex and neonatal lethality. Decreased neuronal proliferation and hypoxia was evident at E11.5, leading to increased neuronal apoptosis in the cortex by E15.5. In order to address whether the observed changes in the structural organization of the nervous system were due to a direct and autocrine role of VEGF-A on the neural population, we conditionally inactivated the main VEGF-A receptor, Flk1, specifically in neuronal lineages, by using the Nestin Cre transgene. The normality of these mice ruled out the possibility that VEGF-A/Flk1 signaling has a significant autocrine role in CNS development. VEGF-A dosage is therefore a critical parameter regulating the density of the vascular plexus in the developing CNS that is in turn a key determinant in the development and architectural organization of the nervous system. Keywords: VEGF-A; Cre recombinase; Conditional gene inactivation; Blood vessel; Endothelium; Nervous system; Cortex; Retina

The Mash2 gene, which encodes a basic helix-loop-helix transcription factor, is one of the mammalian homologues of the Drosophila achaete-scute genes. It is strongly expressed in diploid trophoblast cells of the postimplantation mouse embryo. Targeted mutagenesis of Mash2 revealed that loss of function results in embryonic lethality at midgestation, due to placental failure associated with a lack of spongiotrophoblast and reduced labyrinthine trophoblast layers. For the further study of Mash2 function in development of the trophoblast cell lineage, we have performed chimeric analysis combining Mash2 mutant and wild-type embryos. We have addressed the question of whether the phenotype of the Mash2 mutant embryo, which affects all of the three trophoblast cell layers, is caused by a cell autonomous or non-autonomous defect and whether Mash2 is required in both spongiotrophoblast and labyrinthine trophoblast development. Our results showed no contribution of Mash2 mutant cells to the spongiotrophoblast layer in chimeric placentae at 10.5 and 12.5 days postcoitum, suggesting that the product of the Mash2 gene is required cell autonomously during the development of the spongiotrophoblast. However, it seems that Mash2 is not required for development of labyrinthine trophoblast or giant cells, since high contributions of Mash2 mutant cells were observed in those trophoblast cell layers in the chimeric placentae analyzed. We can therefore conclude that the primary and cell-autonomous function of Mash2 appears to be an involvement in the development of diploid trophoblast cells in the ectoplacental cone to form the spongiotrophoblast cell layer of the mature chorioallantoic placenta.

The possible role of the murine regulator of telomere length (Rtel) gene in telomere length regulation is examined by disrupting its expression in vitro and in vivo. Regulator of telomere length (Rtel) is an essential gene regulating telomere length and prevents genetic instability.

Redox interventions have been controversial in the management of chronic disease. The key reason is believed to be a lack of clarity in our understanding of how endogenous dynamics unfold in biochemical redox mechanisms in live cells. Time-resolved, quantitative research strategies combined with high throughput analysis tools may result in realistic characterisation of related in vivo processes. Here we review new evidence about redox dynamics in live cells. We discuss a potential of this line of research to establish new and affordable ways of redox interventions which may efficiently decrease mortality related to largely preventable chronic diseases. [ABSTRACT FROM AUTHOR]

Zinc is a trace element widely known for its marked antioxidant properties. To gain more insight into the site- and time- specific mechanisms by which it induces chemoprevention, this study was elaborated over a pre-cancerous model of colon carcinogenesis. Colon cancer was induced by 1,2-dimethylhydrazine (DMH) in mice (20 mg/kg for 2 weeks) and groups of animals were supplemented with or without zinc sulfate (ZnSO 4 , 200 mg/L) in drinking water for 4, 10 or 14 weeks. Colon tissues were collected for pathological observation, analyzing aberrant crypt (AC) and aberrant crypt foci (ACF) formations, multiplicity and distribution. Similarly, histological assessment and mucin production, as well as oxidative stress markers estimation was performed for the different groups. Results showed a significant increase in ACF and AC numbers, ACF multiplicity and demonstrated stronger distal occurrence than in the proximal after DHM administration. Histopathological analysis presented marked structural alterations and mucin loss in the distal than the proximal colons. A significant increase in myeloperoxidase (MPO), nitric oxide (NO), L-ornithine and malondialdehyde (MDA) levels was observed followed by a significant decrease in antioxidant markers (superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH)). Oral ZnSO 4 supplementation (continuous or partial) induced significant decrease in ACF, AC numbers and multiplicity, restored histological architecture and mucin production, and a significant decrease in proinflammatory markers while it reduced antioxidants to normal levels. From this study, insight was obtained on the use of ZnSO 4 as a chemopreventive agent and shed light on its potential, as a supplement in nutraceutical approaches. [ABSTRACT FROM AUTHOR]

The groundbreaking discovery of the induced pluripotent stem cells in 2006 [1] brought a major paradigm shift in our understanding of the plasticity of differentiated cells. An unprecedented effort has been invested to elucidate the underpinning molecular mechanisms of the dramatic changes in cell properties, using the powerful secondary reprogramming systems (2°RS). This review outlines the key components of the 2°RS, its recent development and applications that have led to a deeper understanding of the ‘magic’ of induced cell fate changes. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF) is an angiogenic factor and a potent stimulator of microvascular permeability. It is a mitogen specific for endothelial cells. The expression of VEGF and its two receptors, Flk-1 and Flt-1, is pivotal for proper formation of blood vessels in embryogenesis as shown by gene-targeting experiments. Interestingly, the loss of even a single allele of VEGF led to embryonic lethality between day E9.5 and day E10.5 in the mouse. To assess the role of VEGF during embryonic development we decided to tag VEGF expression with LacZ, by inserting an IRES (internal ribosome entry site)-LacZ reporter cassette into the 3[prime] untranslated region of the gene. This alteration enabled us to monitor VEGF expression throughout embryonic development at single-cell resolution. [Beta]-Galactosidase expression from the altered VEGF locus was first observed prior to gastrulation and was detectable at all stages of vascular development in the embryo. Later, the specific cellular distribution and the level of VEGF expression indicated its pleiotropic role in development. High expression levels seemed to be associated with vasculogenesis and permeability, whereas lower levels were associated with angiogenesis and cell migration. In addition, we found VEGF expression in a subtype of endothelial cells present in the endocardium. We believe that the LacZ-tagged allele we have generated offers a precise means of detecting VEGF expression under a variety of physiological and pathological conditions. Key Words: vascular endothelial growth factor; endothelial cells; gene targeting; IRES-lacZ knock-in; embryonic development; vasculogenesis; angiogenesis.

The Cre/loxP site-specific recombination system combined with embryonic stem cell-mediated technologies has greatly expanded our capability to address normal and disease development in mammals using genetic approaches. The success of this emerging technology hinges on the production of Cre-expressing transgenic lines that provide cell type-, tissue-, or developmental stage-specific recombination between loxP sites placed in the genome. Here we describe and characterize the production of a double-reporter mouse line that provides a convenient and reliable readout of Cre recombinase activity. Throughout all embryonic and adult stages, the transgenic animal expresses the lacZ reporter gene before Cre-mediated excision occurs. Cre excision, however, removes the lacZ gene, allowing expression of the second reporter, the human alkaline phosphatase gene. This double-reporter transgenic line is able to indicate the occurrence of Cre excision in an extremely widespread manner from early embryonic to adult lineages. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site-specific recombinase system. Key Words: Cre; loxP; transgenic mice; conditional genome alteration; lacZ; alkaline phosphatase; reporter.

Summary: Control of cellular (de)differentiation in a temporal, cell-specific, and exchangeable manner is of paramount importance in the field of reprogramming. Here, we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming, these factors can be replaced by genes of interest—for example, to enhance lineage-directed differentiation—with the use of a trap-coupled RMCE reaction. We show that, similar to ESCs, Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon in vitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes. [ABSTRACT FROM AUTHOR]

Summary: Vascular endothelial growth factor A (VEGF) is highly expressed in adipose tissue. Its role, however, has not been fully elucidated. Here, we reveal the metabolic role of adipose-VEGF by studying mice with deletion (VEGFAdΔ) or doxycycline-inducible overexpression of a VEGF transgene (VEGFAdTg) in the adipose tissue. VEGFAdΔ mice have reduced adipose vascular density and show adipose hypoxia, apoptosis, inflammation, and metabolic defects on a high-fat diet. In contrast, induction of VEGF expression in VEGFAdTg mice leads to increased adipose vasculature and reduced hypoxia. The latter changes are sufficient to counteract an established compromising effect of high-fat diet on the metabolism, indicating that metabolic misbalance is reversible by adipose vessel density increase. Our data clearly show the essential role of VEGF signaling for adequate adipose function. Besides revealing insights into the molecular mechanisms of obesity-related metabolic diseases, this study points to the therapeutic potential of increased adipose angiogenesis. [Copyright &y& Elsevier]

The ground-breaking work of Yamanaka and Thomson showed that forced expression of just four transcription factors can reprogram mouse and human somatic cells to pluripotency, leading to the discovery of the so-called induced pluripotent stem cells (iPSCs). Similar to embryonic stem cells (ESCs), iPSCs have the ability to permanently self-renew and also give rise to multiple cell types once differentiated. These cells opened up the opportunity to develop human disease models in vitro, drug and toxicity screening tools, as well as a continuous autologous cell source for future cell-based therapies. Therefore, it is not surprising that the methods for generating iPSCs have significantly evolved over the past few years. To date the reprogramming methods include the use of various transfection/transduction systems, small molecules to enhance the reprogramming process, and to adapt to a multitude of different cell type sources. We are now able to convert essentially any somatic cell type into iPSCs with increased efficiency and at higher quality when compared to ESCs. More recently, this field has been expanded to direct reprogramming of one cell type to another, including lineage-specific progenitors. Here, we provide a concise review of methods to generate induced pluripotent stem cells, and discuss the most recent strategies augmenting the reprogramming process and increasing the quality of iPSCs. [Copyright &y& Elsevier]

Summary: Glycogen synthase kinase 3 comprises two isoforms (GSK-3α and GSK-3β) that are implicated in type II diabetes, neurodegeneration, and cancer. GSK-3 activity is elevated in human and rodent models of diabetes, and various GSK-3 inhibitors improve glucose tolerance and insulin sensitivity in rodent models of obesity and diabetes. Here, we report the generation of mice lacking GSK-3α. Unlike GSK-3β mutants, which die before birth, GSK-3α knockout (GSK-3α KO) animals are viable but display enhanced glucose and insulin sensitivity accompanied by reduced fat mass. Fasted and glucose-stimulated hepatic glycogen content was enhanced in GSK-3α KO mice, whereas muscle glycogen was unaltered. Insulin-stimulated protein kinase B (PKB/Akt) and GSK-3β phosphorylation was higher in GSK-3α KO livers compared to wild-type littermates, and IRS-1 expression was markedly increased. We conclude that GSK-3 isoforms exhibit tissue-specific physiological functions and that GSK-3α KO mice are insulin sensitive, reinforcing the potential of GSK-3 as a therapeutic target for type II diabetes. [Copyright &y& Elsevier]

Cardiovascular disease is the leading cause of death for both men and women in North America. Endothelial dysfunction plays a major part in the pathogenesis and progression of vascular disease. Therapeutic interventions that deliver vascular endothelial growth factor (VEGF) to hypoxic tissues have shown great promise in patients with cardiac or peripheral vascular disease by enabling the growth of new blood vessels. By contrast, production of VEGF by solid tumours induces neovascularization required for tumour growth. Given these results, it is no surprise that VEGF has received great attention in the fight against cancer. Currently, there are 33 active clinical trials using anti-VEGF approaches in attempts to cure this disease. The VEGF gene (GenBank accession number NM_003376; gene ID: 7422) is complex and produces numerous isoforms that carry different biological properties. Understanding the mechanism(s) of production, storage, delivery and function of VEGF is fundamental if we are to use this factor effectively in treating patients. [Copyright &y& Elsevier]

Hematopoietic stem and progenitor cell (HSPC) engraftment after transplantation during anticancer treatment depends on support from the recipient bone marrow (BM) microenvironment. Here, by studying physiological homing of fetal HSPCs, we show the critical requirement of balanced local crosstalk within the skeletal niche for successful HSPC settlement in BM. Transgene-induced overproduction of vascular endothelial growth factor (VEGF) by osteoprogenitor cells elicits stromal and endothelial hyperactivation, profoundly impacting the stromal-vessel interface and vascular architecture. Concomitantly, HSPC homing and survival are drastically impaired. Transcriptome profiling, flow cytometry, and high-resolution imaging indicate alterations in perivascular and endothelial cell characteristics, vascular function and cellular metabolism, associated with increased oxidative stress within the VEGF-enriched BM environment. Thus, developmental HSPC homing to bone is controlled by local stromal-vascular integrity and the oxidative-metabolic status of the recipient milieu. Interestingly, irradiation of adult mice also induces stromal VEGF expression and similar osteo-angiogenic niche changes, underscoring that our findings may contribute targets for improving stem cell therapies. [Display omitted] • Establishment of BM hematopoiesis is coupled to development of the skeletal niches • Primary HSPC seeding of bone depends on balanced molecular crosstalk in the niche • Stromal VEGF triggers EC activation and controls stromal-vascular niche integrity • Excessive skeletal VEGF deranges cell metabolism and induces oxidative stress in BM Mesnieres et al. show that increased VEGF in fetal bone disrupts stromal-vascular integrity, alters cell metabolism, and induces oxidative stress in the developing bone marrow niche, blocking the homing of endogenous hematopoietic stem/progenitor cells. Post-natal irradiation, a common stem cell transplantation pretreatment, induces similar niche alterations, thus opening therapeutic strategies. [ABSTRACT FROM AUTHOR]

An abstract of the article "Murine Embryonic Stem Cells," by Andras Nagy and Kristina Vintersten is presented.

The Xp22.11 locus that encompasses PTCHD1 , DDX53 , and the long noncoding RNA PTCHD1-AS is frequently disrupted in male subjects with autism spectrum disorder (ASD), but the functional consequences of these genetic risk factors for ASD are unknown. To evaluate the functional consequences of PTCHD1 locus deletions, we generated induced pluripotent stem cells (iPSCs) from unaffected control subjects and 3 subjects with ASD with microdeletions affecting PTCHD1-AS / PTCHD1 , PTCHD1-AS / DDX53 , or PTCHD1-AS alone. Function of iPSC-derived cortical neurons was assessed using molecular approaches and electrophysiology. We also compiled novel and known genetic variants of the PTCHD1 locus to explore the roles of PTCHD1 and PTCHD1-AS in genetic risk for ASD and other neurodevelopmental disorders. Finally, genome editing was used to explore the functional consequences of deleting a single conserved exon of PTCHD1-AS. iPSC-derived neurons from subjects with ASD exhibited reduced miniature excitatory postsynaptic current frequency and N -methyl-D-aspartate receptor hypofunction. We found that 35 ASD-associated deletions mapping to the PTCHD1 locus disrupted exons of PTCHD1-AS. We also found a novel ASD-associated deletion of PTCHD1-AS exon 3 and showed that exon 3 loss altered PTCHD1-AS splicing without affecting expression of the neighboring PTCHD1 coding gene. Finally, targeted disruption of PTCHD1-AS exon 3 recapitulated diminished miniature excitatory postsynaptic current frequency, supporting a role for the long noncoding RNA in the etiology of ASD. Our genetic findings provide strong evidence that PTCHD1-AS deletions are risk factors for ASD, and human iPSC-derived neurons implicate these deletions in the neurophysiology of excitatory synapses and in ASD-associated synaptic impairment. [ABSTRACT FROM AUTHOR]

Abstract Ischemic stroke results in a loss of neurons for which there are no available clinical strategies to stimulate regeneration. While preclinical studies have demonstrated that functional recovery can be obtained by transplanting an exogenous source of neural progenitors into the brain, it remains unknown at which stage of neuronal maturity cells will provide the most benefit. We investigated the role of neuronal maturity on cell survival, differentiation, and long-term sensorimotor recovery in stroke-injured rats using a population of human cortically-specified neuroepithelial progenitor cells (cNEPs) delivered in a biocompatible, bioresorbable hyaluronan/methylcellulose hydrogel. We demonstrate that transplantation of immature cNEPs result in the greatest tissue and functional repair, relative to transplantation of more mature neurons. The transplantation process itself resulted in the least cell death and phenotypic changes in the immature cNEPs, and the greatest acute cell death in the mature cells. The latter negatively impacted host tissue and negated any potential positive effects associated with cell maturity and the hydrogel vehicle, which itself showed some functional and tissue benefit. Moreover, we show that more mature cell populations are drastically altered during the transplantation process itself. The phenotype of the cells before and after transplantation had an enormous impact on their survival and the consequent tissue and behavioral response, emphasizing the importance of characterizing injected cells in transplantation studies more broadly. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

The purpose of the study described here was to evaluate the usefulness of the elastographic strain ratio in the assessment of liver changes in an experimental animal setting and the hepatoprotective effects of chitosan. Ultrasonography and Strain Ratio calculation were performed before and after bile duct ligation (BDL) in three groups of Wistar albino rats (n = 10 animals per group): (i) rats subjected to bile duct ligation only; (ii) rats subjected to bile duct ligation and administered chitosan for 14 d; (iii) rats subjected to bile duct ligation and administered chitosan for 7 d. The results were compared with the laboratory data and pathologic findings. Strain ratios revealed an increase in liver stiffness after bile duct ligation ( p < 0.05), except in the group with chitosan administered for 7 d, and agreed with laboratory and pathology data. In conclusion, strain ratio can be used as an experimental research instrument in the assessment of liver response to injury. To the best of our knowledge, this is the first study reporting on the usefulness of the sonoelastographic liver-to-kidney strain ratio in assessing the effects of experimentally induced liver lesions. [ABSTRACT FROM AUTHOR]

Summary: Two vascular growth factor families, VEGF and the angiopoietins, play critical and coordinate roles in tumor progression and metastasis. A single inhibitor targeting both VEGF and angiopoietins is not available. Here, we developed a chimeric decoy receptor, namely double anti-angiogenic protein (DAAP), which can simultaneously bind VEGF-A and angiopoietins, blocking their actions. Compared to VEGF-Trap or Tie2-Fc, which block either VEGF-A or angiopoietins alone, we believe DAAP is a highly effective molecule for regressing tumor angiogenesis and metastasis in implanted and spontaneous solid tumors; it can also effectively reduce ascites formation and vascular leakage in an ovarian carcinoma model. Thus, simultaneous blockade of VEGF-A and angiopoietins with DAAP is an effective therapeutic strategy for blocking tumor angiogenesis, metastasis, and vascular leakage. [ABSTRACT FROM AUTHOR]

Interaction of rapidly evolving molecules imparts species specificity to sperm-egg recognition in marine invertebrates, but it is unclear whether comparable interactions occur during fertilization in any vertebrate species. In mammals, the sperm acrosomal protein zonadhesin is a rapidly evolving molecule with species-specific binding activity for the egg zona pellucida (ZP). Here we show using null mice produced by targeted disruption of Zan that zonadhesin confers species specificity to sperm-ZP adhesion. Sperm capacitation selectively exposed a partial von Willebrand D domain of mouse zonadhesin on the surface of living, motile cells. Antibodies to the exposed domain inhibited adhesion of wild-type spermatozoa to the mouse ZP but did not inhibit adhesion of spermatozoa lacking zonadhesin. Zan-/- males were fertile, and their spermatozoa readily fertilized mouse eggs in vitro. Remarkably, however, loss of zonadhesin increased adhesion of mouse spermatozoa to pig, cow, and rabbit ZP but not mouse ZP. We conclude that zonadhesin mediates species-specific ZP adhesion, and Zan-/- males are fertile because their spermatozoa retain adhesion capability that is not species-specific. Mammalian sperm-ZP adhesion is therefore molecularly robust, and species-specific egg recognition by a protein in the sperm acrosome is conserved between invertebrates and vertebrates, even though the adhesion molecules themselves are unrelated. [ABSTRACT FROM AUTHOR]

Abstract: Krüppel-like factor 4 (Klf4, GKLF) was originally characterized as a zinc finger transcription factor essential for terminal differentiation and cell lineage allocation of several cell types in the mouse. Mice lacking Klf4 die postnatally within hours due to impaired skin barrier function and subsequent dehydration. Recently, KLF4 was also used in cooperation with other transcription factors to reprogram differentiated cells to pluripotent embryonic stem cell-like cells. Moreover, involvement in oncogenesis was also ascribed to KLF4, which is aberrantly expressed in some types of tumors such as breast, gastric and colon cancer. We previously have shown that Klf4 is strongly expressed in postmeiotic germ cells of mouse and human testes suggesting a role for Klf4 also during spermiogenesis. In order to analyze its function we deleted Klf4 in germ cells using the Cre–loxP system. Homologous recombination of the Klf4 locus has been confirmed by genomic southern blotting and the absence of the protein in germ cells was demonstrated by Western blotting and immunofluorescence. Despite its important roles in several significant biological settings, deletion of Klf4 in germ cells did not impair spermiogenesis. Histologically, the mutant testes appeared normal and the mice were fertile. In order to identify genes that were regulated by KLF4 in male germ cells we performed microarray analyses using a whole genome array. We identified many genes exhibiting changed expression in mutants even including the telomerase reverse transcriptase mRNA, which is a stem cell marker. However, in summary, the lack of KLF4 alone does not prevent complete spermatogenesis. [Copyright &y& Elsevier]

We demonstrate a novel approach to reverse advanced stages of blindness using hydrogel-mediated delivery of retinal pigmented epithelium (RPE) and photoreceptors directly to the degenerated retina of blind mice. With sodium iodate (NaIO 3) injections in mice, both RPE and photoreceptors degenerate, resulting in complete blindness and recapitulating the advanced retinal degeneration that is often observed in humans. We observed vision restoration only with co-transplantation of RPE and photoreceptors in a hyaluronic acid-based hydrogel, and not with transplantation of each cell type alone as determined with optokinetic head tracking and light avoidance assays. Both RPE and photoreceptors survived significantly better when co-transplanted than in their respective single cell type controls. While others have pursued transplantation of one of either RPE or photoreceptors, we demonstrate the importance of transplanting both cell types with a minimally-invasive hydrogel for vision repair in a degenerative disease model of the retina. Image 1 • A mouse model of severe blindness was used to test a novel cell therapy approach. • An injectable hydrogel was used to deliver cells through a fine needle. • The co-delivery of RPE and photoreceptors led to visual recovery. • The new approach improved transplanted cell survival. [ABSTRACT FROM AUTHOR]