Selectivity is species-dependent: Characterization of standard agonists and antagonists at human, rat, and mouse adenosine receptors.

Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A1, A2A, A2B, and A3, of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A1AR of rat and mouse), CGS-21680 (for A2A AR of rat), and Cl-IB-MECA (for A3AR of all three species). The functionally selective partial A2B agonist BAY60-6583 was found to additionally bind to A1 and A3AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A1), preladenant (A2A), and PSB-603 (A2B) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A3AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.

A Smad3 transgenic reporter reveals TGF-beta control of zebrafish spinal cord development.

TGF-beta (TGFß) family mediated Smad signaling is involved in mesoderm and endoderm specifications, left-right asymmetry formation and neural tube development. The TGFß1/2/3 and Activin/Nodal signal transduction cascades culminate with activation of SMAD2 and/or SMAD3 transcription factors and their overactivation are involved in different pathologies with an inflammatory and/or uncontrolled cell proliferation basis, such as cancer and fibrosis. We have developed a transgenic zebrafish reporter line responsive to Smad3 activity. Through chemical, genetic and molecular approaches we have seen that this transgenic line consistently reproduces in vivo Smad3-mediated TGFß signaling. Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. In the CNS, Smad3 reporter activity is particularly high in the subpallium, tegumentum, cerebellar plate, medulla oblongata and the retina proliferative zone. In the spinal cord, the reporter is activated at the ventricular zone, where neuronal progenitor cells are located. Colocalization methods show in vivo that TGFß signaling is particularly active in neuroD+ precursors. Using neuronal transgenic lines, we observed that TGFß chemical inhibition leads to a decrease of differentiating cells and an increase of proliferation. Similarly, smad3a and 3b knock-down alter neural differentiation showing that both paralogues play a positive role in neural differentiation. EdU proliferation assay and pH3 staining confirmed that Smad3 is mainly active in post-mitotic, non-proliferating cells. In summary, we demonstrate that the Smad3 reporter line allows us to follow in vivo Smad3 transcriptional activity and that Smad3, by controlling neural differentiation, promotes the progenitor to precursor switch allowing neural progenitors to exit cell cycle and differentiate.

Zebrafish reporter lines reveal in vivo signaling pathway activities involved in pancreatic cancer.

Pancreatic adenocarcinoma, one of the worst malignancies of the exocrine pancreas, is a solid tumor with increasing incidence and mortality in industrialized countries. This condition is usually driven by oncogenic KRAS point mutations and evolves into a highly aggressive metastatic carcinoma due to secondary gene mutations and unbalanced expression of genes involved in the specific signaling pathways. To examine in vivo the effects of KRAS(G12D) during pancreatic cancer progression and time correlation with cancer signaling pathway activities, we have generated a zebrafish model of pancreatic adenocarcinoma in which eGFP-KRAS(G12D) expression was specifically driven to the pancreatic tissue by using the GAL4/UAS conditional expression system. Outcrossing the inducible oncogenic KRAS(G12D) line with transgenic zebrafish reporters, harboring specific signaling responsive elements of transcriptional effectors, we were able to follow TGFß, Notch, Bmp and Shh activities during tumor development. Zebrafish transgenic lines expressing eGFP-KRAS(G12D) showed normal exocrine pancreas development until 3 weeks post fertilization (wpf). From 4 to 24 wpf we observed several degrees of acinar lesions, characterized by an increase in mesenchymal cells and mixed acinar/ductal features, followed by progressive bowel and liver infiltrations and, finally, highly aggressive carcinoma. Moreover, live imaging analysis of the exocrine pancreatic tissue revealed an increasing number of KRAS-positive cells and progressive activation of TGFß and Notch pathways. Increase in TGFß, following KRAS(G12D) activation, was confirmed in a concomitant model of medulloblastoma (MDB). Notch and Shh signaling activities during tumor onset were different between MDB and pancreatic adenocarcinoma, indicating a tissue-specific regulation of cell signaling pathways. Moreover, our results show that a living model of pancreatic adenocarcinoma joined with cell signaling reporters is a suitable tool for describing in vivo the signaling cascades and molecular mechanisms involved in tumor development and a potential platform to screen for novel oncostatic drugs.

Generation and application of signaling pathway reporter lines in zebrafish.

In the last years, we have seen the emergence of different tools that have changed the face of biology from a simple modeling level to a more systematic science. The transparent zebrafish embryo is one of the living models in which, after germline transformation with reporter protein-coding genes, specific fluorescent cell populations can be followed at single-cell resolution. The genetically modified embryos, larvae and adults, resulting from the transformation, are individuals in which time lapse analysis, digital imaging quantification, FACS sorting and next-generation sequencing can be performed in specific times and tissues. These multifaceted genetic and cellular approaches have permitted to dissect molecular interactions at the subcellular, intercellular, tissue and whole-animal level, thus allowing integration of cellular and developmental genetics with molecular imaging in the resulting frame of modern biology. In this review, we describe a new step in the zebrafish road to system biology, based on the use of transgenic biosensor animals expressing fluorescent proteins under the control of signaling pathway-responsive cis-elements. In particular, we provide here the rationale and details of this powerful tool, trying to focus on its huge potentialities in basic and applied research, while also discussing limits and potential technological evolutions of this approach.