Fig. 1

Fig. 1

Expression of oncogenic Kras from an astrocyte-specific promoter drives transformation in the larval hindbrain. Live-imaging of mosaic gfap:GFP, gfap:GFP-kras^WT, and gfap:GFP-kras^G12V expression at 3 days post-fertilization (dpf) in the larval zebrafish hindbrain (A). Red dashed outline in schematic of A shows approximate field of view and image orientation for all figures. White dashed lines in representative images denote the hindbrain region of interest in all figures. Morphology of expressing cells was categorized based on characteristics observed throughout the population of injected larvae and larvae were assigned to category of most severe cell morphology identified (B, n = 19 gfap:GFP, 17 gfap:kras^WT, and 38 gfap:kras^G12V larvae; Chi-squared analysis performed). gfap:H2B-mCherry was mosaically co-expressed with gfap:kras plasmids to label nuclei of Kras-expressing cells (C). qRT-PCR was performed on whole-larvae isolates for EMT genes including vimentin, mmp9, cdh1 (e-cadherin), and cdh2 (n-cadherin). Cq were normalized to housekeeping gene rps11 and then normalized to kras^WT condition (D, n = 5 kras^WT and 5 kras^G12V replicates). Larvae were fixed at 3 dpf or 6 dpf and immunostained for N-cadherin (E). N-cadherin co-localization was observed in gfap:kras^G12V-expressing masses at 6 dpf (yellow asterisks). Kras-expressing larvae were fixed at 3 dpf and immunostained for proliferation marker phospho-Histone H3 (pH3, F). pH3/GFP double positive cells were quantified using confocal optical sectioning (single optical slice from yellow dashed box shown in inset) and normalized to the total gfap-expressing area (G, n = 16 larvae/condition). *p < 0.05.