iSuRe-HadCre is an essential tool for effective conditional genetics.

Methods for modifying gene function at high spatiotemporal resolution in mice have revolutionized biomedical research, with Cre-loxP being the most widely used technology. However, the Cre-loxP technology has several drawbacks, including weak activity, leakiness, toxicity, and low reliability of existing Cre-reporters. This is mainly because different genes flanked by loxP sites (floxed) vary widely in their sensitivity to Cre-mediated recombination. Here, we report the generation, validation, and utility of iSuRe-HadCre, a new dual Cre-reporter and deleter mouse line that avoids these drawbacks. iSuRe-HadCre achieves this through a novel inducible dual-recombinase genetic cascade that ensures that cells expressing a fluorescent reporter had only transient Cre activity, that is nonetheless sufficient to effectively delete floxed genes. iSuRe-HadCre worked reliably in all cell types and for the 13 floxed genes tested. This new tool will enable the precise, efficient, and trustworthy analysis of gene function in entire mouse tissues or in single cells.

Notch-mediated cellular interactions between vascular cells.

Vessel formation and differentiation to a proper hierarchical vasculature requires a coordinated effort from endothelial and mural cells. Over the last decade Notch was identified as a key player in this process by promoting vascular arterialization and modulating endothelial tip-stalk phenotypes. Recent work has identified that Notch fine-tunes the diverse endothelial phenotypes through regulation of canonical cell-cycle and metabolism regulators, such as ERK and Myc. During arterialization, Notch signaling inhibits the cell-cycle and metabolism of endothelial cells which coincides with the acquisition of arterial identity. During angiogenesis, the same molecular machinery prevents the hypermitogenic arrest and excessive sprouting of vessels. Notch also signals in pericytes and smooth muscle cells promoting vascular coverage and maturation. Here, we will review the latest findings on how Notch signals regulate the differentiation and interactions among vascular cells during organ development and homeostasis.

Angiocrine polyamine production regulates adiposity.

Reciprocal interactions between endothelial cells (ECs) and adipocytes are fundamental to maintain white adipose tissue (WAT) homeostasis, as illustrated by the activation of angiogenesis upon WAT expansion, a process that is impaired in obesity. However, the molecular mechanisms underlying the crosstalk between ECs and adipocytes remain poorly understood. Here, we show that local production of polyamines in ECs stimulates adipocyte lipolysis and regulates WAT homeostasis in mice. We promote enhanced cell-autonomous angiogenesis by deleting Pten in the murine endothelium. Endothelial Pten loss leads to a WAT-selective phenotype, characterized by reduced body weight and adiposity in pathophysiological conditions. This phenotype stems from enhanced fatty acid ß-oxidation in ECs concomitant with a paracrine lipolytic action on adipocytes, accounting for reduced adiposity. Combined analysis of murine models, isolated ECs and human specimens reveals that WAT lipolysis is mediated by mTORC1-dependent production of polyamines by ECs. Our results indicate that angiocrine metabolic signals are important for WAT homeostasis and organismal metabolism.

Role of Notch in endothelial biology.

The Notch signalling pathway is one of the main regulators of endothelial biology. In the last 20 years the critical function of Notch has been uncovered in the context of angiogenesis, participating in tip-stalk specification, arterial-venous differentiation, vessel stabilization, and maturation processes. Importantly, pharmacological compounds targeting distinct members of the Notch signalling pathway have been used in the clinics for cancer therapy. However, the underlying mechanisms that support the variety of outcomes triggered by Notch in apparently opposite contexts such as angiogenesis and vascular homeostasis remain unknown. In recent years, advances in -omics technologies together with mosaic analysis and high molecular, cellular and temporal resolution studies have allowed a better understanding of the mechanisms driven by the Notch signalling pathway in different endothelial contexts. In this review we will focus on the main findings that revisit the role of Notch signalling in vascular biology. We will also discuss potential future directions and technologies that will shed light on the puzzling role of Notch during endothelial growth and homeostasis. Addressing these open questions may allow the improvement and development of therapeutic strategies based on modulation of the Notch signalling pathway.

Intricacies of conditional genetics in vascular biology.

PURPOSE OF REVIEW: Conditional or inducible recombinase-based genetics is still the gold standard to analyse gene function, given its high specificity, temporal control, limited toxicity and the many available genetic tools. However, it is based on methods that have inherent limitations and shortcomings. The purpose of this review is to summarize and contrast the different available methods used to perform conditional gene function analysis to better inform the community about their particularities and the need to use better methods. RECENT FINDINGS: As any other biomedical field, the vascular biology field has moved from using and analysing standard gene knockout (KO) mice, to use conditional genetics to delete a given gene only at a given time point, cell-type or organ of interest. This is the only way to accurately understand a gene function and avoid other confounding factors. Therefore, nowadays the majority of laboratories working with mice use CreERT2-tamoxifen-inducible genetics. However, this necessary transition from the relatively simple KO genetics to the more sophisticated conditional genetics brought a series of additional methodological issues that are often overlooked or unappreciated. Recent findings from several laboratories have shown how important is to know what to expect from and control for in conditional genetics. Without this a priori knowledge, the quality, robustness, time and costs of conditional genetic experiments can be significantly compromised. SUMMARY: We start this review by discussing the intricacies of the most simple and widely used methods to perform conditional genetics and then extend on the need of novel and more advanced methods to increase the ease, efficiency and reliability of conditional mutagenesis and gene function analysis.

Arterialization requires the timely suppression of cell growth.

The formation of arteries is thought to occur by the induction of a highly conserved arterial genetic programme in a subset of vessels that will later experience an increase in oxygenated blood flow1,2. The initial steps of arterial specification require both the VEGF and Notch signalling pathways3-5. Here, we combine inducible genetic mosaics and transcriptomics to modulate and define the function of these signalling pathways in cell proliferation, arteriovenous differentiation and mobilization. We show that endothelial cells with high levels of VEGF or Notch signalling are intrinsically biased to mobilize and form arteries; however, they are not genetically pre-determined, and can also form veins. Mechanistically, we found that increased levels of VEGF and Notch signalling in pre-arterial capillaries suppresses MYC-dependent metabolic and cell-cycle activities, and promotes the incorporation of endothelial cells into arteries. Mosaic lineage-tracing studies showed that endothelial cells that lack the Notch-RBPJ transcriptional activator complex rarely form arteries; however, these cells regained the ability to form arteries when the function of MYC was suppressed. Thus, the development of arteries does not require the direct induction of a Notch-dependent arterial differentiation programme, but instead depends on the timely suppression of endothelial cell-cycle progression and metabolism, a process that precedes arterial mobilization and complete differentiation.

Endothelial sprouting, proliferation, or senescence: tipping the balance from physiology to pathology.

Therapeutic modulation of vascular cell proliferation and migration is essential for the effective inhibition of angiogenesis in cancer or its induction in cardiovascular disease. The general view is that an increase in vascular growth factor levels or mitogenic stimulation is beneficial for angiogenesis, since it leads to an increase in both endothelial proliferation and sprouting. However, several recent studies showed that an increase in mitogenic stimuli can also lead to the arrest of angiogenesis. This is due to the existence of intrinsic signaling feedback loops and cell cycle checkpoints that work in synchrony to maintain a balance between endothelial proliferation and sprouting. This balance is tightly and effectively regulated during tissue growth and is often deregulated or impaired in disease. Most therapeutic strategies used so far to promote vascular growth simply increase mitogenic stimuli, without taking into account its deleterious effects on this balance and on vascular cells. Here, we review the main findings on the mechanisms controlling physiological vascular sprouting, proliferation, and senescence and how those mechanisms are often deregulated in acquired or congenital cardiovascular disease leading to a diverse range of pathologies. We also discuss alternative approaches to increase the effectiveness of pro-angiogenic therapies in cardiovascular regenerative medicine.

Genetic Tools to Study Cardiovascular Biology.

Progress in biomedical science is tightly associated with the improvement of methods and genetic tools to manipulate and analyze gene function in mice, the most widely used model organism in biomedical research. The joint effort of numerous individual laboratories and consortiums has contributed to the creation of a large genetic resource that enables scientists to image cells, probe signaling pathways activities, or modify a gene function in any desired cell type or time point, à la carte. However, as these tools significantly increase in number and become more sophisticated, it is more difficult to keep track of each tool's possibilities and understand their advantages and disadvantages. Knowing the best currently available genetic technology to answer a particular biological question is key to reach a higher standard in biomedical research. In this review, we list and discuss the main advantages and disadvantages of available mammalian genetic technology to analyze cardiovascular cell biology at higher cellular and molecular resolution. We start with the most simple and classical genetic approaches and end with the most advanced technology available to fluorescently label cells, conditionally target their genes, image their clonal expansion, and decode their lineages.

Fungal community shifts in soils with varied cover crop treatments and edaphic properties.

Cover cropping is proposed to enhance soil microbial diversity and activity, with cover crop type affecting microbial groups in different ways. We compared fungal community compositions of bulk soils differing by cover crop treatment, season, and edaphic properties in the third year of an organic, conventionally tilled rotation of corn-soybean-wheat planted with winter cover crops. We used Illumina amplicon sequencing fungal assemblages to evaluate effects of nine treatments, each replicated four times, consisting of six single winter cover crop species, a three-species mixture, a six-species mixture, and fallow. Alpha-diversity of fungal communities was not affected by cover crop species identity, function, or diversity. Sampling season influenced community composition as well as genus-level abundances of arbuscular mycorrhizal (AM) fungi. Cover crop mixtures, specifically the three-species mixture, had distinct AM fungal community compositions, while cereal rye and forage radish monocultures had unique Core OTU compositions. Soil texture, pH, permanganate oxidizable carbon, and chemical properties including Cu, and P were important variables in models of fungal OTU distributions across groupings. These results showed how fungal composition and potential functions were shaped by cover crop treatment as well as soil heterogeneity.

Publisher Correction: High mitogenic stimulation arrests angiogenesis.

The original version of this Article contained errors in Fig. 8. In panel a, the labels 'VEGF', 'Notch', 'p21', and 'P-ERK' were inadvertently omitted. This has been corrected in the PDF and HTML versions of the Article.

High mitogenic stimulation arrests angiogenesis.

Appropriate therapeutic modulation of endothelial proliferation and sprouting is essential for the effective inhibition of angiogenesis in cancer or its induction in cardiovascular disease. The current view is that an increase in growth factor concentration, and the resulting mitogenic activity, increases both endothelial proliferation and sprouting. Here, we modulate mitogenic stimuli in different vascular contexts by interfering with the function of the VEGF and Notch signalling pathways at high spatiotemporal resolution in vivo. Contrary to the prevailing view, our results indicate that high mitogenic stimulation induced by VEGF, or Notch inhibition, arrests the proliferation of angiogenic vessels. This is due to the existence of a bell-shaped dose-response to VEGF and MAPK activity that is counteracted by Notch and p21, determining whether endothelial cells sprout, proliferate, or become quiescent. The identified mechanism should be considered to achieve optimal therapeutic modulation of angiogenesis.

Data supporting the cover crops benefits related to soil functionality in a 10-year cropping system.

In this data article we provide different field parameters of an agricultural irrigated system under Mediterranean conditions. These parameters represent the response of variables related to soil functionality to different cover crops. Soil and plant samples were taken from fallow and cover crops treatments over the course of 10 years, with most variables measured every other year. This ample database provides reliable information to design sustainable agricultural practices under Mediterranean conditions. Researchers, policy makers and farmers are interested in the final outcome of this dataset. The data are associated with the research article entitled "Cover crops to mitigate soil degradation and enhance soil functionality in irrigated land" (García-González et al., 2018) [1].

Dual ifgMosaic: A Versatile Method for Multispectral and Combinatorial Mosaic Gene-Function Analysis.

Improved methods for manipulating and analyzing gene function have provided a better understanding of how genes work during organ development and disease. Inducible functional genetic mosaics can be extraordinarily useful in the study of biological systems; however, this experimental approach is still rarely used in vertebrates. This is mainly due to technical difficulties in the assembly of large DNA constructs carrying multiple genes and regulatory elements and their targeting to the genome. In addition, mosaic phenotypic analysis, unlike classical single gene-function analysis, requires clear labeling and detection of multiple cell clones in the same tissue. Here, we describe several methods for the rapid generation of transgenic or gene-targeted mice and embryonic stem (ES) cell lines containing all the necessary elements for inducible, fluorescent, and functional genetic mosaic (ifgMosaic) analysis. This technology enables the interrogation of multiple and combinatorial gene function with high temporal and cellular resolution.