Developing a Novel Transgenic Zebrafish Model of Urinary Tract Cancer

Urinary tract cancers encompass cancers of the kidneys, ureters, bladder and urethra. These cancers are common and associated with substantial morbidity and mortality worldwide. The most common of these cancers overall are urothelial carcinomas which arise from the specialised epithelium (termed the urothelium) lining the urinary tract from the renal pelvis down to the urethra, but most commonly affect the bladder. The second most common of these cancers is renal cell carcinoma, an adenocarcinoma, which accounts for approximately 90% of kidney cancers and arises from renal tubular epithelial cells. Large scale sequencing projects have led to important insights on common genomic and transcriptomic alterations in these cancers. However, there is a lack of high volume in-vivo transgenic models of urinary tract cancers available for rigorous mechanistic testing of the roles of these genetic alterations. Zebrafish transgenic models of cancers have demonstrated advantages with respect to ease of genetic manipulation, in-vivo imaging and high-throughput approaches.

I performed a characterisation of the zebrafish excretory urinary tract with respect to histological characteristics and anatomical structure and demonstrate several similarities to the human system. The zebrafish kidneys converge onto two mesonephric ducts (homologous to ureters) which drain into a single saccular epithelium lined urinary bladder. The urinary bladder, in turn, drains externally via a single distinct urethra that is separate from the rectum. The zebrafish urinary epithelium expresses similar proteins to the human system. In addition, I demonstrate the development of the zebrafish urinary bladder with respect to morphological change during the larval and juvenile stages from a tubular structure that develops into a saccular expansile structure at around 6 weeks post fertilisation.

I outline my work on developing a novel transgenic zebrafish urinary tract cancer model using genomic alterations associated with bladder cancer (combination of oncogenic tissue specific HRAS G12V overexpression and a global tp53 DNA binding domain mutation). This model utilises the bipartite Gal4/UAS system and generates cancers in the F0 generation. The cancers are generated by one cell stage microinjection of a plasmid containing the coding sequence for a GFP- HRAS G12V fusion protein (p-UAS: GFP-HRAS_G12V) into a Gal4 enhancer trap zebrafish line (with and without a global tp53 DNA binding domain mutation). We show that this pipeline results in abdominal cancers as early as 3 weeks post-fertilisation (7.1% to 14.6% abdominal tumours between 2 weeks and 3 months for HRASG12V only and combination of tp53 mutation and HRASG12V genomic alterations, respectively). Histological characterisation of these cancers demonstrated them to be situated in the retroperitoneal region of the urinary tract and immunohistochemistry demonstrated them to be a combination of epithelial and non-epithelial in nature. Subsequent attempts at optimisation of this urinary tract cancer model with a more tissue specific zebrafish promoter sequence (cdh17 gene promoter) without using the Gal4/UAS system does not lead to cancers up to 3 months of age suggesting that the Gal4/UAS system is important for cancer formation in zebrafish.