Su Guo

par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed

The first cleavage of C. elegans is asymmetric, generating daughter cells with different sizes, cytoplasmic components, and fates. Mutations in the par-1 gene disrupt this asymmetry. We report here that par-1 encodes a putative Ser/Thr kinase with similarity to kinases from yeasts and mammals. Two strong alleles have mutations in the kinase domain, suggesting that kinase activity is essential for par-1 function. PAR-1 protein is localized to the posterior periphery of the zygote and is distributed in a polar fashion preceding the asymmetric divisions of the germline lineage. Because PAR-1 distribution in the germline correlates with the distribution of germline-specific P granules, it is possible that PAR-1 functions in germline development as well as in establishing embryonic polarity.

Drinks like a fish: zebra fish (Danio rerio) as a behavior genetic model to study alcohol effects

Zebra fish may be an ideal vertebrate model system for numerous human diseases with which the genetics and biological mechanisms of the disease may be studied. Zebra fish has been successfully used in developmental genetics, and recently, neurobiologists have also started to study this species. A potentially interesting target disease amenable for analysis with zebra fish is drug addiction, e.g. alcoholism. Although genetic tools to manipulate the genome of zebra fish are available, appropriate phenotypical testing methods are often lacking. In this paper, we describe basic behavioral tests to investigate the acute effects of alcohol on zebra fish. These behavioral paradigms will be useful for the genetic and biological analysis of acute and chronic drug effects as well as addiction. In addition to presenting findings for the acute effects of alcohol, we briefly describe our strategy for generating and screening mutants. We hope that our pilot work will facilitate the future development of behavioral tests and the use of zebra fish in the genetic analysis of the biological effects of drugs of abuse.

Asymmetrically distributed PAR-3 protein contributes to cell polarity and spindle alignment in early C. elegans embryos

The par-3 gene is required for establishing polarity in early C. elegans embryos. Embryos from par-3 homozygous mothers show defects in segregation of cytoplasmic determinants and in positioning of the early cleavage spindles. We report here that the PAR-3 protein is asymmetrically distributed at the periphery of the zygote and asymmetrically dividing blastomeres of the germline lineage. The PAR-3 distribution is roughly the reciprocal of PAR-1, another protein required for establishing embryonic polarity in C. elegans. Analysis of the distribution of PAR-3 and PAR-1 in other par mutants reveals that par-2 activity is required for proper localization of PAR-3 and that PAR-3 is required for proper localization of PAR-1. In addition, the distribution of the PAR-3 protein correlates with differences in cleavage spindle orientation and suggests a mechanism by which PAR-3 contributes to control of cleavage pattern.

A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion

Initiation and maintenance of infection by mycobacteria in susceptible hosts are not well understood. A screen of Mycobacterium marinum transposon mutant library led to isolation of eight mutants that failed to cause haemolysis, all of which had transposon insertions in genes homologous to a region between Rv3866 and Rv3881c in Mycobacterium tuberculosis, which encompasses RD1 (Rv3871–Rv3879c), a known virulence gene cluster. The M. marinum mutants showed decreased virulence in vivo and failed to secrete ESAT‐6, like M. tuberculosis RD1 mutants. M. marinum mutants in genes homologous to Rv3866‐Rv3868 also failed to accumulate intracellular ESAT‐6, suggesting a possible role for those genes in synthesis or stability of the protein. These transposon mutants and an ESAT‐6/CFP‐10 deletion mutant all showed reduced cytolysis and cytotoxicity to macrophages and significantly decreased intracellular growth at late stages of the infection only when the cells were infected at low multiplicity of infection, suggesting a defect in spreading. Direct evidence for cell‐to‐cell spread by wild‐type M. marinum was obtained by microscopic detection in macrophage and epithelial monolayers, but the mutants all were defective in this assay. Expression of M. tuberculosis homologues complemented the corresponding M. marinum mutants, emphasizing the functional similarities between M. tuberculosis and M. marinum genes in this region that we designate extRD1 (extended RD1). We suggest that diminished membranolytic activity and defective spreading is a mechanism for the attenuation of the extRD1 mutants. These results extend recent findings on the genomic boundaries and functions of M. tuberculosis RD1 and establish a molecular cellular basis for the role that extRD1 plays in mycobacterial virulence. Disruption of the M. marinum homologue of Rv3881c, not previously implicated in virulence, led to a much more attenuated phenotype in macrophages and in vivo, suggesting that this gene plays additional roles in M. marinum survival in the host.

Linking genes to brain, behavior and neurological diseases: what can we learn from zebrafish?

How our brain is wired and subsequently generates functional output, ranging from sensing and locomotion to emotion, decision‐making and learning and memory, remains poorly understood. Dys‐regulation of these processes can lead to neurodegenerative, as well as neuro‐psychiatric, disorders. Molecular genetic together with behavioral analyses in model organisms identify genes involved in the formation of neuronal circuits, the execution of behavior and mechanisms involved in neuro‐pathogenesis. In this review I will discuss the current progress and future potential for study in a newly established vertebrate model organism for genetics, the zebrafish Danio rerio. Where available, schemes and results of genetic screens will be reviewed concerning the sensory, motor and neuromodulatory monoamine systems. Genetic analyses in zebrafish have the potential to provide important insights into the relationship between genes, neuronal circuits and behavior in normal as well as diseased states.

Development of Noradrenergic Neurons in the Zebrafish Hindbrain Requires BMP, FGF8, and the Homeodomain Protein Soulless/Phox2a

We report that the zebrafish mutation soulless, in which the development of locus coeruleus (LC) noradrenergic (NA) neurons failed to occur, disrupts the homeodomain protein Phox2a. Phox2a is not only necessary but also sufficient to induce Phox2b+ dopamine-beta-hydroxylase+ and tyrosine hydroxylase+ NA neurons in ectopic locations. Phox2a is first detected in LC progenitors in the dorsal anterior hindbrain, and its expression there is dependent on FGF8 from the mid/hindbrain boundary and on optimal concentrations of BMP signal from the epidermal ectoderm/future dorsal neural plate junction. These findings suggest that Phox2a coordinates the specification of LC in part through the induction of Phox2b and in response to cooperating signals that operate along the mediolateral and anteroposterior axes of the neural plate.

Acute effects of alcohol on larval zebrafish: a genetic system for large-scale screening

Larval zebrafish are used extensively for developmental genetic studies due to their salient features, such as small size, external development, optical transparency, and accessibility in large numbers. However, their use for the study of drug and alcohol abuse has not been explored. Here we investigated the response of larval zebrafish to acute treatment of alcohol. Our analyses showed that like adults, the larval zebrafish exhibited a dose-dependent locomotor response to ethanol: intermediate doses led to hyperactivity, whereas high doses have a neurodepressive effect resulting in hypoactivity and sedation. Alcohol also induced morphological changes of melanocytes, providing a visible cellular measure of the biological effects of alcohol in vivo. In addition, alcohol induced thigmotaxis behavior (preference for the edge of a compartment). In the behaviors we analyzed, genetic background influenced the locomotor responses to alcohol. The present study demonstrates that larval zebrafish exert a response to the acute treatment of alcohol, which is genetically modifiable. Therefore, the larval zebrafish represent a tractable vertebrate model system for a large-scale genetic analysis of the biological effects of alcohol.

Molecular genetics of asymmetric cleavage in the early Caenorhabditis elegans embryo.

Asymmetric cleavage plays an important role in Caenorhabditis elegans embryogenesis. In addition to generating cellular diversity, several early asymmetric cleavages contribute to the spatial organization of the embryo. Genetic and molecular analyses of several genes, including six par genes and the mex-1 and mes-1 genes, together with experimental embryological studies, have provided insights into mechanisms controlling polarity and spindle orientations during these cleavages. In particular, these studies focus attention on microfilament-based motility and changing protein distributions at the cell cortex.

A regulator of transcriptional elongation controls vertebrate neuronal development.

The development of distinct vertebrate neurons is defined by the unique profiles of genes that neurons express. It is accepted that neural genes are regulated at the point of transcription initiation, but the role of messenger RNA elongation in neural gene regulation has not been examined. Here we describe the mutant foggy, identified in a genetic screen for mutations that affect neuronal development in zebrafish, that displayed a reduction of dopamine-containing neurons and a corresponding surplus of serotonin-containing neurons in the hypothalamus. Positional cloning disclosed that Foggy is a brain-enriched nuclear protein that is structurally related to the transcription elongation factor Spt5 (refs 5,6,7,8,9,10,11 ,12). Foggy is not part of the basic transcription apparatus but a phosphorylation-dependent, dual regulator of transcription elongation. The mutation disrupts its repressive but not its stimulatory activity. Our results provide molecular, genetic and biochemical evidence that negative regulators of transcription elongation control key aspects of neuronal development.

The PINK1/Parkin pathway regulates mitochondrial dynamics and function in mammalian hippocampal and dopaminergic neurons

PTEN-induced putative kinase 1 (PINK1) and Parkin act in a common pathway to regulate mitochondrial dynamics, the involvement of which in the pathogenesis of Parkinsons disease (PD) is increasingly being appreciated. However, how the PINK1/Parkin pathway influences mitochondrial function is not well understood, and the exact role of this pathway in controlling mitochondrial dynamics remains controversial. Here we used mammalian primary neurons to examine the function of the PINK1/Parkin pathway in regulating mitochondrial dynamics and function. In rat hippocampal neurons, PINK1 or Parkin overexpression resulted in increased mitochondrial number, smaller mitochondrial size and reduced mitochondrial occupancy of neuronal processes, suggesting that the balance of mitochondrial fission/fusion dynamics is tipped toward more fission. Conversely, inactivation of PINK1 resulted in elongated mitochondria, indicating that the balance of mitochondrial fission/fusion dynamics is tipped toward more fusion. Furthermore, overexpression of the fission protein Drp1 (dynamin-related protein 1) or knocking down of the fusion protein OPA1 (optical atrophy 1) suppressed PINK1 RNAi-induced mitochondrial morphological defect, and overexpression of PINK1 or Parkin suppressed the elongated mitochondria phenotype caused by Drp1 RNAi. Functionally, PINK1 knockdown and overexpression had opposite effects on dendritic spine formation and neuronal vulnerability to excitotoxicity. Finally, we found that PINK1/Parkin similarly influenced mitochondrial dynamics in rat midbrain dopaminergic neurons. These results, together with previous findings in Drosophila dopaminergic neurons, indicate that the PINK1/Parkin pathway plays conserved roles in regulating neuronal mitochondrial dynamics and function.