Project: Daughterless Cane toad
I am currently working on a novel invasive species control
technology called “Daughterless” This new strategy of
control has only been possible in recent times due to the
advancement of molecular biology techniques and discoveries.
Essentially, the daughterless project involves the discovery of a
“male determining” gene in cane toads and creating a
transgenic toad which will carry this gene, effectively causing all
carrier animals to develop as males, regardless of their male, or
female genotype. Additionally, this transgene, since it is
incorporated into the genome of the animals, will be passed on to
all offspring in future generations. Once a daughterless toad is
created, it will be released into the wild to spread it’s
“daughterless gene” throughout the population, bring
about a shortage of females and eventually causing the population
to collapse.
Project outline:
Due to the considerable lack of understanding of cane toad sex
determination, I have chosen to take a multi-pronged approach to
this project. My approach involves cytology as well as basic
molecular biology to tease out potential sex determining genes in
the cane toads.
The cytological aspect of my project is geared towards the
discovery of sex chromosomes in cane toads. Toads have
traditionally been thought to have a ZZ/ZW sex chromosome system
(as opposed to the more familiar XX/XY system of mammals). This
change in nomenclature is due to the fact that the female of the
species has two different chromosomes, while the male has two of
the same. This work was done at Australia National University in
collaboration with Dr. Jennifer Graves and Dr. Tariq Ezaz. We
were able to successfully identify the potential “W”
chromosome of cane toads using modern techniques not available
previously in the field. This discovery will greatly aid in
narrowing a potential gene or chromosomal locus which is
responsible for sex determination in this species.
The second approach I decided to take in investigating the sex
determination of cane toads is to clone “candidate” sex
determining genes with close orthologs in the mammalian system.
From previous studies on a variety of animals, we know that the sex
determination pathway among vertebrates is fairly well conserved
with regards to key genes. I cloned six of these key genes ( Sox9,
Wt1, Dmrt1, p450, Sf1, and Dax1) in order to analyse their function
in the developing cane toads. Thus far I have complete expressed
sequences from Sox9, Dmrt1 and p450, as well as partial sequences
for the other three. Furthermore, I have real time expression data
showing the expression of these key genes in the testis and ovaries
of developing cane toads, ranging from pre-sexual differentiation
stage to adults.
Additionally, I have been working on a novel theory in sex
determination in this species. All male and female toads carry what
is thought to be a rudimentary ovary called a Bidder’s organ
as a key defining characteristic in the clade. By using the cloned
genes, I was able to assess the expression level of these key genes
in this organ to find some interesting expression profiles which I
plan to follow in the coming year of my PhD. I will test the role
of this organ as a potential developmental aid in sex
determination.
