Background
Diffuse midline gliomas (DMGs) are paediatric brain tumours that affect over 2,000 children in Europe and the United States every year1. These tumours are typically diagnosed in the brainstem and less often in the midbrain, spine, and thalamus with clinical symptoms such as facial asymmetry, dysarthria, ataxia, decreased strength and hyperreflexia. Patients have very high mortality, with the median survival being only 9-11 months, and 90% of children die within two years of diagnosis. Chemotherapy is the standard treatment used for DMGs2.
The increased availability of tumour tissue through diagnostic biopsies and post-mortem tumour specimen donations, coupled with the advancement in genomic profiling, has facilitated an increased molecular understanding of this fatal disease3–5. The genomic landscape of DMGs reveals a somatic hotspot mutation in a histone H3 isoform (eg, H3.3K27M) and about 20 additional driver mutations (eg, TP53 in 70% of H3.3K27M-mutant patients, and ACVR1 in 80% of H3.1K27M-mutant patients)1,3,6–8. Several studies have suggested oligodendrocyte progenitor cells (OPC) and neural stem/progenitor cells (NSC) as the likely cell of origin of H3K27M-mutant DMGs5,9–11. However, it is currently unclear when during brain development, initial somatic mutations arise in DMGs.
Zebrafish (Danio rerio) has become a popular model organism in oncology12,13. Over 70% of human genes have orthologues in the zebrafish genome, and the percentage goes up to 82% if one considers disease-related genes14. Zebrafish cancer models can further be used to investigate the origin of tumour cells, tumour cell migration, proliferation, and microenvironment interactions15–17.
The laboratory is currently generating transgenic H3.3K27M-mutant zebrafish lines expressed under different promoters representing different cell types. Therefore, we seek a highly motivated master’s student who would help characterise these lines at single-cell resolution.
Project description
The project's objective is to characterise three genetically engineered zebrafish models driven under the control of promoters thought to be the cellular origin of H3K27M DMGs at different developmental stages. This will serve as a reference atlas for comparing the transcriptomic changes that occur during tumourigenesis and predicting which promoter-expressing cell populations give rise to DMG tumours in the subsequent experiments involving promoter-specific expression of the H3K27M mutation.
Methods
The methods utilised in this project include zebrafish engineering, genotyping, fluorescence-activated cell sorting (FACS), fluorescent imaging, mRNA isolation, RT-qPCR, and single-cell transcriptomics with the 10x Genomics technology.
Research Environment and Supervision
The project will be carried out at the Computational Oncology Group at the Centre for Molecular Medicine Norway (NCMM) with office/lab space at the Forksningsparken (https://waszaklab.org). The students will be supervised by Dr Nancy Saana Banono (NCMM, project supervisor), Prof Sebastian Martin Waszak (NCMM, main supervisor), and Prof Sandra Lopez Avi