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. RESULTS: Our survey revealed several similarities, e.g., main endpoints of home cage and openfield observations, species, and positive control substances, but also a high level of heterogeneity between different companies with regard to behavioral endpoints during handling and reflex testing, scoring, group size, and timing of studies. Analysis of heterogeneously designed historical studies with amphetamine and chlorpromazine showed the anticipated behavioral changes, albeit with quantitative variability, and identified more robust (e.g., activity, posture, muscle tone, startle reflex, body temperature) and less robust (piloerection, stereotypical behavior, palpebral closure, respiration) Irwin/FOB parameters. A partially standardized interlaboratory test with MK-801 and chlorpromazine showed the expected behavioral changes and principally confirmed the historically-based more/less robust Irwin/FOB parameters, however, it also showed exposure variability and did not show a markedly reduced quantitative variability of behavioral results. DISCUSSION: Our survey and intercompany test results demonstrate certain heterogeneity in design and conduct of Irwin/FOB tests by pharmaceutical companies. Although the general behavioral profiles for the reference compounds were consistently found, quantitative variability of results remained even under partially standardized conditions. This suggests the importance of a high level of standardization with regard to the Irwin/FOB test modification used, scoring system, and observer training, in order to achieve an improved intercompany comparability of Irwin/FOB results.

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). wnt4b expression in the medial and lateral floor plate are shown to be regulated independently: medial floor plate expression occurs in the absence of a notochord, while lateral floor plate expression requires a functional notochord, sonic hedgehog and gli-2.

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. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

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. The loss of cranial motor neurons is not due to aberrant hindbrain patterning, failure of neurogenesis, increased cell death or absence of hh expression. Furthermore, activation of the Hh pathway, which normally induces branchiomotor neurons, fails to induce motor neurons in the dtr hindbrain. Despite this, not all Hh-mediated regulation of hindbrain development is abolished since the regulation of a neural gene by Hh is intact in the dtr hindbrain. Finally, dtr can function cell autonomously to induce branchiomotor neurons. These results suggest that detour encodes a component of the Hh signaling pathway that is essential for the induction of motor neurons in the hindbrain but not in the spinal cord and that dtr function is required for the induction of only a subset of Hh-mediated events in the hindbrain.

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. Our results support the idea that most u-type genes are required for Hedgehog signalling and indicate that while such signalling is essential for slow myocyte differentiation, the loss of activity of one signal, Sonic hedgehog, can be partially compensated for by other Hedgehog family proteins.

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. These results suggest that Shh and/or Twhh play an important role in the induction of branchiomotor neurons in vivo. In sonic-you (syu) mutants, where Shh activity was reduced or eliminated due to mutations in shh, branchiomotor neurons were reduced in number in a rhombomere-specific fashion, but never eliminated. Similarly, spinal motor neurons were reduced, but not eliminated, in syu mutants. These results demonstrate that Shh is not solely responsible for inducing branchiomotor and spinal motor neurons, and suggest that Shh and Twhh may function as partially redundant signals for motor neuron induction in zebrafish.

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. In the myotome, but not the neural tube, netrin-1a expression requires sonic hedgehog. In embryos lacking sonic hedgehog, the sonic-you locus, netrin-1a expression is reduced or absent in the myotomes but present in the neural tube. Embryos lacking sonic hedgehog express tiggy-winkle hedgehog in the floor plate, suggesting that, in the neural tube, tiggy-winkle hedgehog can compensate for the lack of sonic hedgehog in inducing netrin-1a expression. Ectopic expression of sonic hedgehog, tiggy-winkle hedgehog, or echidna hedgehog induces ectopic netrin-1a expression in the neural tube, and ectopic expression of sonic hedgehog or tiggy-winkle hedgehog, but not echidna hedgehog, induces ectopic netrin-1a expression in somites. These data demonstrate that in vertebrates netrin expression is regulated by Hedgehog signaling.

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. In contrast to mouse embryos lacking Shh activity, syu mutant embryos do form medial floor plate cells and motorneurons. Since ectopic overexpression of shh in zebrafish embryos does not induce ectopic medial floor plate cells, we conclude that shh is neither required nor sufficient to induce this cell type in the zebrafish.

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. The pipetail mutant phenotype is characterized by defects in tail formation and impaired maturation of the cells that contribute to cartilaginous elements of the head skeleton. This suggests a major role for wnt5 in morphogenetic processes underlying tail outgrowth and cartilage differentiation in the head. To investigate the function of maternally derived wnt5 mRNA, we generated females that were homozygous for pipetail. The lack of a maternal effect phenotype in the progeny of these females suggests that no obvious function for the maternal wnt5 expression can be deduced.

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. The protein translated from the shorter mRNA does not show this difference. This indicates that Drosophila like vertebrates has two forms of gelsolin, one secreted, the other cytoplasmic. The mRNA for both is present ubiquitously in the early embryo. Later, the cytoplasmic form is expressed in parts of the gut. The RNA for the secreted form is expressed in the fat body, and the secreted protein is abundant in extracellular fluid (hemolymph). The cytoplasmic form of gelsolin co-localizes with F-actin in the cortex of the cells in the embryo and in larval epithelia. However, during cellularization of the blastoderm it is reduced at the base of the cleavage furrow, a structure similar to the contractile ring in dividing cells.