RESEARCH OBJECTIVES

The research of the group falls into two categories that are complementary to each other, but find common ground in the use of zebrafish to provide a model of perturbed gene expression. I should stress here that our research uses molecular genetic tools to identify gene expression outcomes, and not necessarily phenotypic outcomes. In this context, the research should be viewed in the light of providing a different slant on the use of zebrafish outside the confines of a developmental biology tool.


Functional Genomics

Functional genomics involves the development and application of global experimental approaches to assess gene function by making use of the information and reagents provided by genome sequencing and mapping. In this context, we have embarked on a dual strategy for effecting changes in gene expression in the zebrafish in order to assess the consequences of these changes at a global level. These strategies involve targeted mutagenesis as well as gene down-regulation to model inherited human disorders, with an emphasis on Duchenne muscular dystrophy (DMD). This neuromuscular disorder is due to mutations in the muscle protein, dystrophin, which cause loss of dystrophin at the muscle membrane surface and destabilisation of a multi-protein dystrophin-associated glycoprotein complex. We propose that modelling in the zebrafish offers a better means of assessing the real-time dynamics of gene expression and protein localisation during muscle development compared to mouse studies.

Our experimental approach is essentially two-fold. First, to undertake the selective mutagenesis of the zebrafish genes by introducing targeted peptide nucleic acids (PNAs). Secondly, to achieve transient gene down-regulation using PNAs targeted to splice sites in order to effect out-of-frame mutation events, as well as RNA interference. Each of these strategies is presented below.

Gene targeting reagents: Triplex-forming oligonucleotides, side-by-side minor groove binders, and helix-invading peptide nucleic acids (PNAs) are effective as gene-specific transcriptional blocking agents. The latter reagent is stable against nucleases and proteases, binds independently of salt concentration and, due to its neutral backbone, has a much higher affinity for nucleic acids than do DNA/DNA duplexes. In addition, modified PNAs offer the possibility of not only blocking transcription, but of introducing mutations in the targeted region during DNA replication. Several groups have reported introducing mutations in mammalian cells and yeast using psoralen-conjugated oligonucleotides. Psoralen is a bifunctional photoreagent that introduces a covalent crosslink into target sequences following irradiation at 365nm. We are currently using a number of modified PNAs, including psoralen-conjugated PNAs, as part of a collaborative project that addresses the targeting of zebrafish genes. In the first instance, we are targeting the zebrafish tyrosinase gene. If this approach proves efficient then we will concentrate on targeting genes implicated in human disease processes. The aim here is to introduce not only nonsense mutations but more subtle missense mutations in functionally critical domains of disease-causing proteins, thus improving our understanding of disease processes in the context of global gene expression rather than a single gene:phenotype relationship; the use of microarrays will be the cornerstone of this research.

Translational knock-down: RNA interference (RNAi), which involves the use of double-stranded (ds) RNA, has been successfully applied to knockdown the expression of specific genes in plants, D. melanogaster, C. elegans, trypanosomes, planaria, hydra, and several vertebrate species including the mouse and zebrafish. As a functional genomic tool, RNAi has great potential for the functional analysis of uncharacterised genes and has already been widely applied to high throughput screens in C. elegans and has recently been shown to be effective in cultured mammalian cells in the form of short interfering RNAs.
Several studies have reported non-specific effects following the direct introduction of dsRNA into zebrafish. However, these effects may simply be due to the injection of large quantities of dsRNA into the developing embryo during a period of precisely controlled expression of a vast array of vertebrate developmental genes. In this respect, neither the use of dsRNA-producing vectors, nor an examination of the effects of RNAi on global gene expression, has been undertaken in the zebrafish. Our current research is directed to addressing these two concerns.
As an ancillary aspect of transient gene down-regulation, we are also looking at the effects of PNAs targeted to splice sites as effectors of out-of-frame mutation events. We have concentrated our work here on the zebrafish dystrophin gene and the effects of perturbed dystrophin expression on the glycoprotein complex with which dystrophin interacts.

The above projects attempt to replicate autosomal recessive-type human diseases. In an attempt to examine autosomal dominant-type diseases, we are currently looking at the modelling of Huntington Disease as well as a rare bone disorder in the zebrafish. We have constructed a variety of recombinants that carry expanded polyglutamine repeats under the control of a constitutive promoter, as well as the zebrafish HD gene promoter. The effect of expression of these recombinants on endogenous gene expression in the zebrafish will be examined using microarrays. In terms of bone disorders, we have examined the RANK-RANK-L/OPG pathway in the zebrafish and are currently constructing several OPG mutants that we have identified in patients with idiopathic hyperphosphatasia. These constructs will be introduced into zebrafish embryos to assess the impact of aberrant OPG expression on zebrafish skeleton formation.


Chemical Genomics

The identification of new medicines and the development of therapeutics involve the investigation and exploration of defined molecule interactions with complex biological processes. This specificity of action can provide drug prototypes and involves the modulation of gene product function, which comprises the area of Chemical Genomics. This field has been enhanced recently by studying the effects of drug prototypes on not only pure protein targets, but also on an organism’s global network of protein interactions. This type of analysis has involved the development of whole-organism gene expression microarrays in which an organism's entire protein-coding potential (expressed sequences) is spotted onto glass slides in high-density arrays. The slides are subsequently hybridised using fluorescently labelled reverse-transcribed RNA and the slides scanned to detect fluorescent signal. The analysis of these signals provides a measure of expression levels of the genes that are represented on the array. To date, this type of work has been undertaken using invertebrate species only, but has overlooked the use of zebrafish as a model of vertebrate development.

In terms of those compounds that have been studied in the context of chemical genomics, a range of novel and structurally diverse natural products has been isolated from marine organisms (Ascidians and Sponges) collected from shallow water sites around the coast of New Zealand. Such metabolites include terpenes, sulfur-containing alkaloids, purine derivatives and amino-acid derived compounds. Many marine natural products exhibit wide ranges of pharmaceutically interesting biological activities, such as antitumour, antiviral and antimicrobial properties. However, some natural products lack activity in these assays, which are limited in scope and fail to dissect the effect of compounds on complex biological processes. We are interested in these natural products as they may collectively hold the key to providing valuable information about cellular function and development while offering a new biological resource for medicines and therapeutics.
The purpose of this project is to develop the zebrafish as a robust analytical platform for drug discovery by assessing the effects of chemicals and drugs on global gene expression in developing vertebrate embryos. A necessary part of this research is to develop the zebrafish as a high-throughput bioassay system for the in vivo analysis of the effects of natural and synthetic compounds. This bioassay system lends itself to the dissection of biological processes, while also offering the means of assessing the effects of chemicals on disease processes.
The zebrafish is one of two vertebrate models of human development as well as disease, and this species offers several advantages over that of the mouse. However, to take advantage of the favourable features of the zebrafish, further development of an effective means of directed disease modelling as well as assessing the pleiotropic effects of perturbed gene expression are required. In the case of disease modelling, our group is investigating a promising approach of combining gene-specific peptide nucleic acid (PNA) mutagenesis, together with transient inhibition of, or down-regulation of genes encoding, DNA repair proteins. However, the context of this research aim, and the opportunity of embracing the development of a novel chemical genomics platform requires proof-of-principle, and it is here that this research proposal is concerned.
We have designed a zebrafish microarray that targets apparent orthologues of genes known to be involved in the apoptosis pathway. This type of array will allow us to use an antisense approach involving PNAs to key genes in the apoptosis pathway, as well as alternative chemical inhibitors that are available. The efficiency and effect of transient gene down regulation effected by PNAs can then be directly compared to chemical inhibition of the gene product by using gene expression microarray analysis.

The apoptosis pathway has also been selected as it is a key component of over 26 human diseases and disorders as well as being critical for normal development. Apoptosis pathways will be characterised throughout the developing zebrafish to help identify key genes involved in specific developmental stages and to provide the first description of the specific apoptotic genes involved in each developmental stage. The effects of small chemical inhibitors on specific genes involved in apoptosis will facilitate the molecular dissection of this important pathway. We will then design small chemical or PNA inhibitors for genes involved in the Huntington Disease neurodegenerative pathway and assess the impact of these inhibitors on the onset and progression of this disease and measure secondary effects of cellular toxicity by using microarray analysis. We will take advantage of the conclusion of the public genome sequencing of the zebrafish, scheduled for late 2002, to incorporate additional disease-related genes into our array with view of making the first commercially available zebrafish microarray targeting disease processes. We intend to demonstrate the utility of zebrafish as a high-throughput bioassay system by screening a range of structurally diverse compounds isolated from marine organisms collected from around New Zealand against our microarray. Since only a small number of chemical inhibitors are currently available, we hope to detect compounds that may be of commercial interest, either as potential therapeutic reagents or for functional genomics analysis.


Microarrays

The School of Biological Sciences has invested in a Stanford-type microarray platform for the arraying of cDNAs and oligonucleotides. An Axon 4000B GenePix scanner and associated software support the arrayer in our attempts to screen for the effects of perturbed gene expression in the zebrafish brought about by DNA-based as well as chemical reagents.