Figure 1. CCDC6-RET and NCOA4-RET directed cell migration and whole animal lethality in Drosophila.
A. Schematic of the full length, wild-type RET gene and the two RET fusions CCDC6-RET and NCOA4-RET.
B. Histogram quantifying the percent larvae that matured to pupariation vs. balancer controls (Ctrl: tub>w-/Tubby, CC6-R: tub>CCDC6-RET/Tubby, NC4-R: tub>NCOA4-RET/Tubby). Animals expressing low levels of RET fusions under control of the tubulin-Gal4 driver died during larval stages. GAL4 activity—and therefore expression levels—were controlled by temperature. tub>CCDC6-RET was expressed at a higher level (27°C) yet led to only 42% larval lethality; tub>NCOA4-RET was expressed at lower levels (25°C) and led to 100% larval lethality. Data are represented as mean +/− SEM. Asterisks: ** p ≤ 0.01, **** p ≤ 0.0001.
C. Entire third instar larval wing disc expressing GFP in the ptc expression domain. Perimeter of wing is outlined to show shape of the disc. Region shown in D–F″ shown in yellow rectangle. Scale bar represents 315μm.
D–F″. Late, third instar larval wing discs expressing transgenes in the ptc expression domains as indicated; transformed cells were visualized with UAS-GFP. D′–F′. Localization with a phospho-RET antibody confirmed that human RET fusions are expressed and activated in Drosophila tissue. D″–F″. Merged panels highlight that only those cells expressing a RET fusion transgene activated RET. Scale bar in F represents 50 μm in D–F″.
G–I. Z-stack images of previously described wing discs showing migrating cells. Control animals (F) show no migration from endogenous ptc-GAL4 expression domain, while ptc>CCDC6-RET (G) and ptc>NCOA4-RET (H) have one or more cells that have left the original space and migrated into wild-type neighboring tissue; brackets show the distance cells have traveled from the posterior edge of the ptc domain and white arrows indicate cells that have migrated. Apical membrane is marked in blue with DE-Cadherin staining, emphasizing that migrating cells have lost their polarity to invade along the basal membrane. Scale bar in G represents 16.7μm in G–I.
J. Quantification of the severity of cell migration caused by each RET fusion expression under control of the ptc-GAL4 driver in larval wing discs. Wing discs were scored blind and binned into one of four categories (severe, moderate, weak, or no migration). Example images for each category is provided in Fig. S2. Again despite differences in temperature, more ptc>CCDC6-RET (27°C) was expressed in the wing disc and less ptc>NCOA4-RET (25°C), yet ptc>NCOA4-RET displayed a significantly more severe phenotype. Data are represented as mean +/− SEM. Asterisks: **** p ≤ 0.0001.
See also Figure S2