A zebrafish sarcoma shelf screen elucidates PAX3-FOXO1 cell lineage tolerance and transforma on capacity

Genevieve C. Kendall1,2, Colle e A. LaVigne1,2, Whitney Murchison1,2, Dinesh Rakheja1,3, Koichi Kawakami4, James F. Amatruda1,2,5

1. Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX USA.
2. Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX USA.
3. Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX USA.
4. Division of Molecular and Developmental Biology, Na onal Ins tute of Gene cs, Mishima, Japan.
5. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX USA.

Rhabdomyosarcoma is the most common pediatric so ssue cancer and displays characteris cs of developing skeletal muscle. Of the two major histological subtypes, embryonal and alveolar (ARMS), ARMS is a more aggressive disease with a higher rate of metastasis. The chromosomal transloca ons t(1;13) or t(2;13) gene cally characterize the majority of ARMS, resul ng in either PAX3 or PAX7 fused to FOXO1 to create a transcrip onally ac ve fusion protein. The presence of PAX3-FOXO1 correlates to reduced overall survival in pa ents, yet the mechanisms by which tumorigenesis is ini ated, relevant cell lineages, and the pathways targeted have not been fully described. We u lized the UAS/GAL4 and Tol2 system in order to develop vertebrate models of PAX3- FOXO1 ARMS with cell lineage targeted expression. First, we injected UAS:PAX3-FOXO1 directly into 26 GAL4 transgenic zebrafish lines with ssue- speci c GAL4 expression. PAX3- FOXO1 inhibited somitogenesis or induced cyclopia in targeted cells. Relevant lines with embryonic phenotypes were further evaluated for their u lity as models for studying ARMS by crossing with a stable transgenic UAS:mCherry2A- PAX3- FOXO1 zebrafish line generated and validated by our group. To date, six GAL4 lines have been tested as part of an ongoing e ort, with two crosses yielding o spring with detectable PAX3- FOXO1 expression. Many progeny from one of these crosses failed to develop in ated swim bladders and did not survive past one month. Expression of PAX3-FOXO1 in the second GAL4 line cross was de ned by diminished eye pigmenta on at 48 hours post fer liza on with 40% of these sh developing tumors in their eyes by 50 days of age. Our curated GAL4 line collec on cons tutes a shelf screen for rhabdomyosarcoma and other sarcomas, allowing us to test variegated expression of PAX3-FOXO1 and providing a comprehensive func onal valida on of the ac vity of gene fusions in a vertebrate system.