A systematic assessment of robustness in CNS safety pharmacology.

Background And Purpose: Irwin tests are key preclinical study elements for characterising drug-induced neurological side effects. This multicentre study aimed to assess the robustness of Irwin tests across multinational sites during three stages of protocol harmonisation. The projects were part of the Enhanced Quality in Preclinical Data framework, aiming to increase success rates in transition from preclinical testing to clinical application.

Variability of non-clinical behavioral CNS safety assessment: An intercompany comparison.

Introduction: Irwin/FOB testing is routinely conducted to investigate the neurofunctional integrity of laboratory animals during preclinical development of new drugs, however, the study design frequently varies to meet specific needs. Representatives of several European-based pharmaceutical companies performed a "state-of-the-art" assessment of how they conduct their CNS safety evaluation using Irwin/FOB tests.

Methods: This assessment consisted of (1) a survey of current/historical practice, (2) an evaluation of historical studies with reference compounds (amphetamine, chlorpromazine) to determine intercompany reproducibility of results, and (3) an interlaboratory test using reference compounds (MK-801, chlorpromazine) to determine whether partially standardized conditions (animals, sex, doses, vehicles, administration route, observation time points, systemic exposure) might reduce variability of results.

Discovery of BI 135585, an in vivo efficacious oxazinanone-based 11β hydroxysteroid dehydrogenase type 1 inhibitor.

A potent, in vivo efficacious 11β hydroxysteroid dehydrogenase type 1 (11β HSD1) inhibitor (11j) has been identified. Compound 11j inhibited 11β HSD1 activity in human adipocytes with an IC of 4.3nM and in primary human adipose tissue with an IC of 53nM.

Large-scale mapping of mutations affecting zebrafish development.

Background: Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers.

iguana encodes a novel zinc-finger protein with coiled-coil domains essential for Hedgehog signal transduction in the zebrafish embryo.

Signaling by lipid-modified secreted glycoproteins of the Hedgehog family play fundamental roles during pattern formation in animal development and in humans; dysfunction of Hedgehog pathway components is frequently associated with a variety of congenital abnormalities and cancer. Transcriptional regulation of Hedgehog target genes is mediated by members of the Gli zinc-finger transcription factors. The relative nuclear concentrations of Gli activator (Gli(act)) and repressor (Gli(rep)) forms, together with their nucleocytoplasmic trafficking, appear to be critical determinants for target gene expression.

Zebrafish wnt4b expression in the floor plate is altered in sonic hedgehog and gli-2 mutants.

The floor plate of the neural tube serves an important function as a source of signals that pattern cell fates in the nervous system as well as directing proper axon pathfinding. We have cloned a novel zebrafish wnt family member, wnt4b, which is expressed exclusively in the floor plate. To place wnt4b in the context of known regulators of midline development, its expression was analyzed in the zebrafish mutants cyclops (cyc), floating head (flh), you-too (yot), and sonic you (syu).

Control of muscle cell-type specification in the zebrafish embryo by Hedgehog signalling.

The specification of different muscle cell types in the zebrafish embryo requires signals that emanate from the axial mesoderm. In previous studies we and others have shown that overexpression of different members of the Hedgehog protein family can induce the differentiation of two types of slow-twitch muscles, the superficially located slow-twitch fibres and the medially located muscle pioneer cells. Here we have investigated the requirement for Hedgehog signalling in the specification of these distinct muscle cell types in two ways: first, by characterising the effects on target gene expression and muscle cell differentiation of the u-type mutants, members of a phenotypic group previously implicated in Hedgehog signalling, and second, by analysing the effects of overexpression of the Patched1 protein, a negative regulator of Hedgehog signalling.

A radiation hybrid map of the zebrafish genome.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome.

The zebrafish detour gene is essential for cranial but not spinal motor neuron induction.

The zebrafish detour (dtr) mutation generates a novel neuronal phenotype. In dtr mutants, most cranial motor neurons, especially the branchiomotor, are missing. However, spinal motor neurons are generated normally.

Role of sonic hedgehog in branchiomotor neuron induction in zebrafish.

The role of zebrafish hedgehog genes in branchiomotor neuron development was analyzed by examining mutations that affect the expression of the hedgehog genes and by overexpressing these genes in embryos. In cyclops mutants, reduction in sonic hedgehog (shh) expression, and elimination of tiggy-winkle hedgehog (twhh) expression, correlated with reductions in branchiomotor neuron populations. Furthermore, branchiomotor neurons were restored in cyclops mutants when shh or twhh was overexpressed.

Regulation of netrin-1a expression by hedgehog proteins.

Netrins, a family of growth cone guidance molecules, are expressed both in the ventral neural tube and in subsets of mesodermal cells. In an effort to better understand the regulation of netrins, we examined the expression of netrin-1a in mutant cyclops, no tail, and floating head zebrafish embryos, in which axial midline structures are perturbed. Netrin-1a expression requires signals present in notochord and floor plate cells.

Sonic hedgehog is not required for the induction of medial floor plate cells in the zebrafish.

Sonic hedgehog (Shh) is a secreted protein that is involved in the organization and patterning of several tissues in vertebrates. We show that the zebrafish sonic-you (syu) gene, a member of a group of five genes required for somite patterning, is encoding Shh. Embryos mutant for a deletion of syu display defects in patterning of the somites, the lateral floor plate cells, the pectoral fins, the axons of motorneurons and the retinal ganglion cells.

Wnt5 is required for tail formation in the zebrafish embryo.

Intercellular signaling molecules, such as those encoded by the Wnt gene family, have a fundamental role in various aspects of pattern formation in the developing embryo. The zebrafish wnt5 gene encodes a member of a subfamily of Wnt molecules thought to be involved in modulating cell behavior during vertebrate development. Here, we show that the zebrafish pipetail gene is identical to wnt5.

Identification of secreted and cytosolic gelsolin in Drosophila.

We have cloned the gene for Drosophila gelsolin. Two mRNAs are produced from this gene by differential splicing. The protein encoded by the longer mRNA has a signal peptide and its electrophoretic mobility when translated in vitro in the presence of microsomes is higher than when it is translated without microsomes.