Dr Ruth Arkell

Phone (02) 61259158
e-mail: Ruth.Arkell@anu.edu.au

Disorders of forebrain development have a variety of consequences for the affected individuals. As well as intellectual, motor, language, memory and emotive deficits and epilepsy, children with forebrain defects often also exhibit craniofacial abnormalities, agenesis of the olfactory bulbs, poor respiration and neuroendocrinopathies such as Diabetes Insipidus. The genetic and clinical heterogeneity of forebrain defects along with practical and ethical constraints of human embryological studies hinders research in this field using human subjects. An alternative approach is to use mouse genetics since, where available, mutations in the homologous mouse genes also cause forebrain defects. These mutations, combined with increasingly powerful mouse genetic tools and resources, make the mouse an excellent model system for analyzing the manner in which the mammalian forebrain develops.

Project 1: Assaying Zic5 function in the developing mouse brain (Dr. Radiya Ali and Dr Ruth Arkell)

Background:
This project focuses specifically on a condition called microcephaly, a neuro-developmental disorder in which a forebrain of normal architecture but reduced size is generated in utero. Microcephaly results from a reduction in the number of neural cells potentially brought about by reduced proliferation or increased cell death of neural progenitors. The decreased brain size is especially evident in the cerebral cortex, the forebrain structure associated with higher cognitive functions, thus correspondingly microcephaly is tightly correlated with mental retardation. We have generated a mouse model of microcephaly by making mutations in the zinc finger containing transcription factor Zic5. We have mouse strains carrying different mutations in Zic5 and our studies indicate that post-translational modification of Zic5 by the addition of a SUMO moiety is essential for Zic5 function. Preliminary data suggest that, once SUMOylated, Zic5 localises to a particular component of the nucleus.

The project:
You will confirm this preliminary data by expressing fluorescent versions of Zic5 and SUMO in mammalian cell lines and use confocal microscopy to demonstrate that they co-localise to specific structures within the nucleus. You will also identify the nuclear structure to which they localise by hybridisation of antibodies that label known structures. In the third part of the project you will analyse the embryonic phenotype of a mouse mutant to determine whether the in vitro disruption of SUMOylation leads to loss of Zic5 function. This will be achieved by dissecting embryos and comparing their features to those of a known null allele of Zic5.

Experience gained: 
This project will give you experience in molecular, biochemical, cell biological and embryological techniques such as bacterial transformation, plasmid purification, culture and transfection of mammalian cells, protein expression, quantification and detection, fluorescence confocal microscopy and mouse embryo dissection.

Project 2: An improved Zic trans-activation assay (Dr. Radiya Ali and Dr Ruth Arkell)

Background
Our laboratory studies the function of a group of transcription factors known as the Zic family. We study the three Zic genes that are expressed during gastrulation (Zic 2, 3 and 5). When each of these is mutated they give rise to a distinct embryonic phenotype: mutation of Zic2 leads to a disorder of forebrain development (holoprosencephaly), mutation of Zic3 causes problems in the establishment of the Left-Right body axis and is accompanied by situs defects in many organs and mutation of Zic5 leads to a brain with normal architecture but reduced size (microcephaly). One tool frequently used to study transcription factors is cell based transactivation assays. In these experiments a DNA binding site is placed upstream of a reporter gene. This construct is co-transfected into mammalian cells with an expression plasmid containing a gene that should bind the DNA binding site and activation of the reporter gene is monitored. Currently, these assays use a luciferase gene as the reporter and activity is detected in a luminometer. The transcativation assay for the Zic genes suffers from the fact that all Zic genes stimulate the reporter well. The aim of this project is to construct a more sensitive trans-activation assay to enable discrimination between Zic family members with respect to their ability to trans-activate the reporter.

The Project
You will construct a plasmid that contains a Zic DNA binding site upstream of a fluorescent reporter known as pTimer and develop methods for quantifying fluorescence following the expression of this construct in mammalian cell lines. You will then compare the behaviour of this construct in trans-activation assays to that of an analogous construct that incorporates a luciferase reporter. In the third part of the project you will use your new trans-activation assay to identify sequences within the divergent Zic2/5 promoter that are stimulated by Zic3 binding and determine whether other Zic family members may also act on these sequences.

Experienced gained
This project will give you experience in molecular, biochemical and cell biological techniques such as plasmid construction using a new restriction enzyme free method of cloning, plasmid transformation, plasmid purification, the culture and transfection of mammalian cells and protein expression, quantification and detection..