Honours supervisors and projects

Dr Heather Verkade

Telephone: 9905 4663
Email: heather.verkade@sci.monash.edu.au

The focus of the laboratory is to understand how genetics controls the complex cell behaviours that are required for organ morphogenesis during development.  We use the model system Danio rerio, the zebrafish, to analyse the cell behaviours during vertebrate development, and we mainly focus on the development of the endodermal organs such as the intestine, liver and pancreas.

During development, cells undergo many changes in behaviour, shape, adhesion and polarity.  These are tightly regulated by signalling pathways and transcription factors, and ultimately determine the position and structure of the embryo’s organs.  These cell behaviours are highly reminiscent of the behaviour that cancer cells acquire during cancer formation and metastasis.  We hope that by understanding the genetic control of cell behaviour during the normal process of embryogenesis, we will be uncovering the genes that could endow cancer cells with the same cell behaviours in the disease state.

We approach these questions using the most powerful techniques available in zebrafish research.  The cell behaviour and embryo development are analysed using wholemount stains such as in situ hybridisation, and the production of fluorescent transgenic lines.  We analyse the shape, polarity and behaviour of individual cells using confocal microscopy and fluorescence immuno-histochemistry.  Several novel mutants are currently being characterised using positional cloning methods including PCR genotyping.

Project 1. Genetic and phenotypic characterization of a zebrafish mutant showing defective gut development

In 2003, I conducted a genetic screen to identify zebrafish mutants that show defective gut morphogenesis. I isolated three interesting mutants. Preliminary phenotypic characterization of these mutants suggests that early endodermal cell movements or cell interactions are disrupted.

In this project, you would carry out a detailed phenotypic characterization of mutant embryos to identify the exact nature of the defects in intestine, liver and pancreas development, and to determine if these mutants show defects in any other (non-endodermal) organs. The analysis will involve the following techniques: setting up fish crosses to collect embryos, in situ hybridisation to observe patterns of gene expression patterns, analysis of the fluorescence patterns in embryos from transgenic backgrounds, and possibly fluorescent immunohistochochemistry of embryo sections to observe cell structure and protein localisation.

In order to identify the genes that are mutated in these mutants, genetic mapping has been started. Heterozygous carriers of the mutation have been crossed to mapping strains, and mapping embryos have been collected.

In this project you would continue the genetic mapping using gel-scorable PCR markers SSLPs (simple sequence length polymorphisms). This mapping forms one part of a larger positional cloning project (which usually takes ~2 years). The following techniques will be used: DNA extraction, PCR, gel electrophoresis, bioinformatics, and possibly sequencing and primer design.

This project contains several techniques, and so can be adjusted to suit your particular interests.

Project 2. Analysis of the role of candidate genes on endodermal cell movements

We use a transgenic strain of zebrafish which expresses green fluorescent protein (GFP) in the endodermal cells during early embryogenesis Tg(sox17:GFP). This allows us to track the moving cells in the living embryo, and thus to characterize these early movements.

Only one pathway has been determined to regulate endodermal cell movement. This is the Wnt/PCP pathway, which controls the polarity of many different types of cells as they move. Migratory cells may to use this pathway to orient themselves relative to the direction of migration. We believe that more genes are likely to be involved in this complex process. We have identified several candidate genes.

In this project you will disrupt the expression of one of these candidate genes in zebrafish embryos using an antisense oligo approach. You do this by injecting wildtype embryos with an antisense oligo called a morpholino. You will then analyse the phenotype of these embryos, looking at the whole structure, and more specifically at the early intestine. If we identify a defect in the formation of the early intestine, you will concentrate on analysis of the movement, shape and structure of the endodermal cells in the antisense treated embryos. The following techniques will be used: setting up fish to collect embryos, morpholino injections, brightfield and fluorescence microscopy and in situ hybridisation.  Further possible analysis techniques would be fluorescence immunohistochemistry of sections and timelapse fluorescence microscopy.

GEN3990 students:  Projects are available that concentrate on just one aspect of development, to fully characterise the defect caused by knockdown of expression of these candidate genes.  Techniques include: setting up fish to collect embryos, morpholino injections, brightfield and fluorescence microscopy and in situ hybridisation. 

Project 3: Zebrafish as a model to study turnover of the mitochondrion by autophagy

In collaboration with Professor Rod Devenish and Dr. Mark Prescott (Biochemistry and Molecular Biology).

The mitochondrion is a key organelle linked to both the life and death of a cell. However, little is known concerning the mechanisms used by the cell to control mitochondrial turnover after damage or during normal course of development. The predominant mechanism for the elimination of mitochondria is believed to be the evolutionarily conserved process of autophagy (or more specifically mitophagy) whereby mitochondria are delivered to lysosomes for degradation and recycling by the cell. Although autophagy is responsible for the turnover of many different cellular components, current evidence suggests mitophagy is regulated in specific manner. We are interested in the mechanism by which individual mitochondria within the cell during development are first recognised then delivered to the lysosome.

Mitophagy in live cells can followed using fluorescent protein technology. This project will involve the use of molecular biology techniques to generate transgenic zebrafish embryos expressing a novel fluorescent biosensor for autophagy targeted to the mitochondrion. Fluorescence microscopy will be used to characterise the behaviour of mitochondria during the early stages of zebrafish development. Subsequent studies would the use of this valuable tool to identify and characterise the role of genes involved in mitophagy during development.