Zebrafish as a model of BRAFV600E melanoma subtypes and Nevus biology

Abstract

The most frequent mutation identified in both benign nevi and malignant melanoma is the constitutively activating V600E substitution of BRAF. However, how additional mutations co-operate with BRAFV600E to promote subtypes of melanoma in animals is only beginning to be understood. In this thesis, I generate and analyze zebrafish BRAFV600E melanoma models and also develop the first animal model for BRAFV600E nevus recurrence.

In my first data chapter, I develop a unique animal model of nevus recurrence. In people it is not uncommon for a nevus to recur following removal, even when no pigmented nevus cells remain. The biology of how and why this happens is unknown. By partial amputation of the nevus in the zebrafish tail fin, we described both nevus regrowth, as well as nevi that do not regrow. Utilising melanin as a lineage tracer, I was able to show that recurrent nevi are repopulated from an unpigmented precursor population. This suggested that BRAFV600E nevi are supported by an undifferentiated stem cell population that is recruited to regenerate and pigment the nevus after removal.

In my second data chapter, I use genetics to develop BRAFV600E zebrafish models of melanoma. In collaboration with Dr. James Lister and Professor Jeroen den Hertog, I describe three differing models of zebrafish melanoma. All three models show progression to melanoma, and in collaboration with Dr. Marie Mathers I establish that while BRAFV600E is present in all three models, co-operating mutations affect melanoma pathology.

In my third data chapter, I develop tools to study the molecular differences in the BRAFV600E melanoma models. I described the optimisation of a broad range of antibodies, raised against human peptides due to the lack of reliable antibodies in the zebrafish field. I use punch core biopsies of both zebrafish and human tumours, and whole sagittal sections of juvenile zebrafish, to show specific staining throughout many organs of the developing fish. I then use some of these antibodies to analyse molecular pathways in the melanoma models.